Methods for preparing modified von willebrand factor

ABSTRACT

The present invention provides modified von Willebrand Factor molecules, methods for their preparation and uses thereof. The invention further provides pharmaceutical compositions for treating coagulation disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/576,061, filed Nov. 21, 2017, which is the U.S. national stage entryunder 35 U.S.C. § 371 of International Application No.PCT/EP2016/061440, filed on May 20, 2016, which claims priority toEuropean Patent Application No. 15168934.6, filed on May 22, 2015. Thecontents of these applications are each incorporated herein by referencein their entirety.

FIELD OF THE INVENTION

The present invention relates to products and methods for improvingtreatment of blood coagulation disorders.

BACKGROUND OF THE INVENTION

There are various bleeding disorders caused by deficiencies of bloodcoagulation factors. The most common disorders are hemophilia A and B,resulting from deficiencies of blood coagulation Factor VIII (FVIII) andIX, respectively. Another known bleeding disorder is von Willebrand'sdisease (VWD).

In plasma FVIII exists mostly as a noncovalent complex with vonWillebrand Factor (VWF), and its coagulant function is to accelerateFactor IXa dependent conversion of Factor X to Xa.

Classic hemophilia or hemophilia A is an inherited bleeding disorder. Itresults from a chromosome X-linked deficiency of blood coagulationFVIII, and affects almost exclusively males with an incidence of betweenone and two individuals per 10,000. The X-chromosome defect istransmitted by female carriers who are not themselves hemophiliacs. Theclinical manifestation of hemophilia A is an increased bleedingtendency.

In severe hemophilia A patients undergoing prophylactic treatment FVIIIhas to be administered intravenously (i.v.) about 3 times per week dueto the short plasma half-life of FVIII of about 12 to 14 hours. Eachi.v. administration is cumbersome, associated with pain and entails therisk of an infection especially as this is mostly done at home by thepatients themselves or by the parents of children having been diagnosedfor hemophilia A.

It would thus be highly desirable to increase the half-life of FVIII sothat pharmaceutical compositions containing such FVIII would have to beadministered less frequently.

Several attempts have been made to prolong the half-life ofnon-activated FVIII either by reducing its interaction with cellularreceptors (WO 03/093313 A2, WO 02/060951 A2), by covalently attachingpolymers to FVIII (WO 94/15625, WO 97/11957 and U.S. Pat. No.4,970,300), by encapsulation of FVIII (WO 99/55306), by introduction ofnovel metal binding sites (WO 97/03193), by covalently attaching the A2domain to the A3 domain either by peptidic (WO 97/40145 and WO03/087355) or disulfide linkage (WO 02/103024A2) or by covalentlyattaching the Al domain to the A2 domain (WO2006/108590).

Another approach to enhance the functional half-life of FVIII or VWF isby PEGylation of FVIII (WO 2007/126808, WO 2006/053299, WO 2004/075923)or by PEGylation of VWF (WO 2006/071801). The increased half-life ofpegylated VWF would indirectly also enhance the half-life of FVIIIpresent in plasma. Also fusion proteins of FVIII have been described (WO2004/101740, W02008/077616 and WO 2009/156137).

VWF, which is missing, functionally defect or only available in reducedquantity in different forms of von Willebrand disease (VWD), is amultimeric adhesive glycoprotein present in the plasma of mammals, whichhas multiple physiological functions. During primary hemostasis VWF actsas a mediator between specific receptors on the platelet surface andcomponents of the extracellular matrix such as collagen. Moreover, VWFserves as a carrier and stabilizing protein for procoagulant FVIII. VWFis synthesized in endothelial cells and megakaryocytes as a 2813 aminoacid precursor molecule. The amino acid sequence and the cDNA sequenceof wild-type VWF are disclosed in Collins et al. 1987, Proc. Natl. Acad.Sci. USA 84:4393-4397. The precursor polypeptide, pre-pro-VWF, consistsof an N-terminal 22-residue signal peptide, followed by a 741-residuepro-peptide and the 2050-residue polypeptide found in mature plasma VWF(Fischer et al., FEBS Lett. 351: 345-348, 1994). After cleavage of thesignal peptide in the endoplasmatic reticulum a C-terminal disulfidebridge is formed between two monomers of VWF. During further transportthrough the secretory pathway 12 N-linked and 10 O-linked carbohydrateside chains are added. More important, VWF dimers are multimerized viaN-terminal disulfide bridges and the propeptide of 741 amino acidslength is cleaved off by the enzyme PACE/furin in the late Golgiapparatus.

Once secreted into plasma the protease ADAMTS13 can cleavehigh-molecular weight VWF multimers within the Al domain of VWF. PlasmaVWF therefore consists of a whole range of multimers ranging from singledimers of 500 kDa to multimers consisting of up to more than 20 dimersof a molecular weight of over 10,000 kDa. The VWF-HMWM hereby having thestrongest hemostatic activity, which can be measured in ristocetincofactor activity (VWF:RCo). The higher the ratio of VWF:RCo/VWFantigen, the higher the relative amount of high molecular weightmultimers.

In plasma FVIII binds with high affinity to VWF, which protects it frompremature elimination and thus, plays in addition to its role in primaryhemostasis a crucial role to stabilize FVIII, regulate plasma levels ofFVIII and as a consequence is also a central factor to control secondaryhemostasis. The half-life of non-activated FVIII bound to VWF is about12 to 14 hours in plasma. In von Willebrand disease type 3, where no oralmost no VWF is present, the half-life of FVIII is only about 2 to 6hours, leading to symptoms of mild to moderate hemophilia A in suchpatients due to decreased concentrations of FVIII. The stabilizingeffect of VWF on FVIII has also been used to aid recombinant expressionof FVIII in CHO cells (Kaufman et al. 1989, Mol Cell Biol 9:1233-1242).

VWF-derived polypeptides, in particular VWF fragments, have beendescribed to stabilize FVIII in vitro and in vivo. WO 2013/106787 Al isdirected at chimeric proteins comprising certain VWF fragments and aFVIII protein. WO 2014/198699 A2 and WO 2013/083858 A2 describe VWFfragments and their use in the treatment of hemophilia. WO 2011/060242A2 discloses fusion polypeptides comprising certain VWF fragments and anantibody Fc region. W02013/093760 A2 describes a method for preparing aprotein, comprising co-expressing FVIII or VWF polypeptides, includingtruncated forms of VWF, with a recombinant α-2,3-sialyltransferase. Yeeet al. (2014) Blood 124(3):445-452 found that a VWF fragment containingthe D′D3 domains is sufficient to stabilize Factor VIII in mice.However, although a VWF D′D3-Fc fusion protein exhibited markedlyprolonged survival when transfused into FVIII-deficient mice, the VWFD′D3-Fc fusion protein did not prolong the survival of co-transfusedFVIII.

The effect of the fermentation temperature on the sialylation level of aglycoprotein was investigated by Trummer et al (Biotech. Bioeng. (2006)Vol. 94, No. 6, p. 1033-1044) who found for erythropoietin at 30° C. adecrease in sialylation by 40% and at 33° C. a decrease by 20%.

Ahn et al. also investigated the effect of fermentation temperature onthe sialylation level of a glycoprotein and published for erythropoietin(Biotech. Bioeng.(2008) Vol. 101, No. 6, p.

1234-1244) a percentage of asialo glycoprotein at 37° C. of 2.4%, and at32° C. of 2.1%.

There is an ongoing need for methods increasing the half-life of FVIIIand FVIII products with reduced administration frequency.

SUMMARY OF THE INVENTION

It has been found by the inventors that the sialylation of N-glycans ofVWF fragments can be significantly increased if mammalian cellstransfected with recombinant DNA encoding a VWF fragment are cultured ata lowered temperature, e.g. below 36° C. The products obtained in thisway exhibit improved pharmacokinetics and a prolonged mean residencetime (MRT) and can be used to also improve pharmacokinetics and prolongMRT of a co-administered FVIII. It has been found by the inventors thatthe clearance of FVIII can be significantly reduced by co-administrationof a half-life extended VWF-derived polypeptide which is characterizedby a high degree of sialylation of its N-glycans. They are thereforeparticularly suitable for treating blood coagulation disorders.Especially VWF fragments capable of binding to FVIII which compriseN-glycans wherein more than 75% of all N-glycans on average have atleast one sialic acid have been shown to be particularly useful.

Another advantage of the method of the present invention as describedabove is that the

VWF fragments obtained have a higher proportion of dimers than VWFfragments produced in a conventional manner. The inventors found thatthe dimers have a higher affinity to FVIII than the monomers.

In particular preferred embodiments of the invention the VWF-derivedpolypeptide of the invention may be connected to a half-life extendingmoiety and is characterized by a high degree of sialylation of itsN-glycans and has a particular low amount of N-glycans with multivalentterminal and non-sialylated galactose residues, including a particularlow amount of N-glycans with two or more terminal and non-sialylatedgalactose residues, and even more preferred a particular low amount ofN-glycans with three or more terminal and non-sialylated galactoseresidues.

The present invention therefore relates to the following embodiments [1]to [53]:

[1] A method of producing a glycoprotein comprising N-glycans withincreased sialylation, which comprises (i) providing cells comprising anucleic acid encoding a polypeptide comprising a truncated vonWillebrand Factor (VWF), and (ii) culturing said cells at a temperatureof less than 36.0° C., wherein said polypeptide comprising a truncatedVWF preferably has a circulatory mean residence time (MRT) greater thanthat of full-length VWF.

[2] A method of producing a dimer of a glycoprotein comprising atruncated von Willebrand Factor (VWF), which comprises (i) providingcells comprising a nucleic acid encoding the amino acid sequence of theglycoprotein, and (ii) culturing said cells at a temperature of lessthan 36.0° C.

[3] A method of increasing the dimerization of a glycoprotein comprisinga truncated von Willebrand Factor (VWF), which comprises (i) providingcells comprising a nucleic acid encoding amino acid sequence of theglycoprotein, and (ii) culturing said cells at a temperature of lessthan 36.0° C.

[4] The method of any one of the preceding items, wherein the cellsfurther comprise a recombinant nucleic acid encoding asialyltransferase, preferably an α-2,6-sialyltransferase and/or anα-2,3-sialyltransferase.

[5] The method of any one of the preceding items, wherein prior to step(ii) the cells are cultured at a temperature of 37.0° C.±1.0° C., andstep (ii) comprises culturing the cells at a temperature of 34.0°C.±2.0° C.

[6] A method of producing a glycoprotein comprising N-glycans withincreased sialylation, which comprises (i) providing cells comprising anucleic acid encoding a polypeptide comprising a truncated vonWillebrand Factor (VWF) and a recombinant nucleic acid encoding anα-2,6-sialyltransferase, and (ii) culturing the cells under conditionsthat allow expression of the glycoprotein.

[7] The method of any one of the preceding items, wherein the cells aretransfected mammalian cells, and step (i) comprises introducing intomammalian cells the nucleic acid encoding a polypeptide comprising thetruncated VWF, and optionally the recombinant nucleic acid encoding asialyltransferase.

[8] The method of any one of the preceding items, further comprising thestep of recovering the glycoprotein from the culture medium.

[9] The method of any one of the preceding items, further comprisingsubjecting the glycoprotein obtained in any one of the preceding itemsto ion exchange chromatography, whereby glycoprotein with highsialylation is separated from glycoprotein with low sialylation; andcollecting the fractions eluted from the ion exchange column having highsialylation.

[10] The method of any one of the preceding items, further comprisingcontacting the glycoprotein obtained in any one of the preceding itemswith a sialyltransferase and a sialic acid donor in vitro.

[11] The method of item [10], wherein the sialyltransferase is anα-2,6-sialyltransferase, an α-2,3-sialyltransferase, or a combinationthereof.

[12] The method of any one of the preceding items, wherein theglycoprotein further comprises a half-life extending heterologouspolypeptide fused to the truncated VWF.

[13] The method of item [12], wherein the half-life extendingheterologous polypeptide comprises or consists of a polypeptide selectedfrom the group consisting of albumin or a fragment thereof having alength of at least 100 amino acids, immunoglobulin constant regions andportions thereof, e.g. the Fc fragment, transferrin and fragmentsthereof, the C-terminal peptide of human chorionic gonadotropin,solvated random chains with large hydrodynamic volume known as XTEN,homo-amino acid repeats (HAP), proline-alanine-serine repeats (PAS),albumin, afamin, alpha-fetoprotein, Vitamin D binding protein,polypeptides capable of binding under physiological conditions toalbumin or immunoglobulin constant regions, and combinations thereof.

[14] The method of any one of items [1] to [11], comprising conjugatingthe glycoprotein obtained in any one of the preceding items with thehalf-life-extending moiety.

[15] The method of item [14], wherein the half-life-extending moiety isselected from the group consisting of hydroxyethyl starch (HES),polyethylene glycol (PEG), polysialic acids (PSAs) and albumin bindingligands, e.g. fatty acid chains.

[16] The method of any one of the preceding items, wherein, on average,at least 75% of the N-glycans of the obtained glycoprotein comprise atleast one sialic acid moiety.

[17] The method of any one of the preceding items, wherein, on average,at least 80% of the N-glycans of the obtained glycoprotein comprise atleast one sialic acid moiety.

[18] The method of any one of the preceding items, wherein, on average,at least 85% of the N-glycans of the obtained glycoprotein comprise atleast one sialic acid moiety.

[19] The method of any one of the preceding items, wherein, on average,at least 50% of the obtained glycoprotein is present as dimer.

[20] A glycoprotein obtainable by a method of any one of the precedingitems.

[21] A glycoprotein comprising a truncated von Willebrand Factor (VWF),wherein said truncated VWF is capable of binding to a Factor VIII(FVIII), and wherein said glycoprotein comprises N-glycans, and at least75%, preferably at least 85%, more preferably at least 90%, preferablyat least 95%, preferably at least 96%, preferably at least 97%,preferably at least 98%, more preferably at least 99% of said N-glycanscomprise, on average, at least one sialic acid moiety.

[22] A glycoprotein comprising a truncated von Willebrand Factor (VWF),wherein said truncated VWF is capable of binding to a Factor VIII(FVIII), and wherein said glycoprotein comprises N-glycans, wherein lessthan 35%, preferably less than 34%, preferably less than 33%, preferablyless than 32%, preferably less than 31%, preferably less than 30%,preferably less than 29%, preferably less than 28%, preferably less than27% preferably less than 26%, preferably less than 25%, preferably lessthan 24%, preferably less than 23%, preferably less than 22%, preferablyless than 21%, preferably less than 20%, preferably less than 19%,preferably less than 18%, preferably less than 17%, preferably less than16%, preferably less than 15%, preferably less than 14%, preferably lessthan 13%, preferably less than 12%, preferably less than 11%, preferablyless than 10%, preferably less than 9%, preferably less than 8%,preferably less than 7%, preferably less than 6% and preferably lessthan 5% of said N-glycans comprise, on average, two or more terminal andnon-sialylated galactose residues.

[23] A glycoprotein according to items [21] and [22].

[24] A glycoprotein comprising a truncated von Willebrand Factor (VWF),wherein said truncated VWF is capable of binding to a Factor VIII(FVIII), and wherein said glycoprotein comprises N-glycans, wherein lessthan 6%, preferably less than 5%, preferably less than 4%, preferablyless than 3%, preferably less than 2%, and preferably less than 1% ofsaid N-glycans comprise, on average, three or more terminal andnon-sialylated galactose residues.

[25] A glycoprotein according to items [21] and [24] or to items [22]and [24] or to items [23] and [24].

[26] The glycoprotein of items [21] to [25], wherein at least 70% ofsaid N-glycans comprise, on average, at least one α-2,6-sialic acidmoiety or one α-2,3-sialic acid moiety.

[27] The glycoprotein of any one of items [20] to [26], wherein thetruncated VWF comprises (a) amino acids 776 to 805 of SEQ ID NO:9 or (b)an amino acid sequence having a sequence identity of at least 90% toamino acids 776 to 805 of SEQ ID NO:9.

[28] The glycoprotein of any one of items [20] to [27], wherein thetruncated VWF comprises (a) amino acids 766 to 864 of SEQ ID NO:9 or (b)an amino acid sequence having a sequence identity of at least 90% toamino acids 766 to 864 of SEQ ID NO:9.

[29] The glycoprotein of any one of items [20] to [28], wherein thetruncated VWF consists of (a) amino acids 764 to 1242 of SEQ ID NO:9,(b) an amino acid sequence having a sequence identity of at least 90% toamino acids 764 to 1242 of SEQ ID NO:9, or (c) a fragment of (a) or (b).

[30] The glycoprotein of any one of items [20] to [29], furthercomprising a half-life extending heterologous polypeptide fused to thetruncated VWF, and/or a half-life-extending moiety conjugated to theglycoprotein.

[31] The glycoprotein of item [30], wherein said half-life extendingheterologous polypeptide comprises or consists of human serum albumin ora fragment thereof, wherein the length of said fragment is at least 100amino acids.

[32] The glycoprotein of item [30], wherein said heterologouspolypeptide fused to the glycoprotein comprises or consists of apolypeptide selected from the group consisting of immunoglobulinconstant regions and portions thereof, e.g. the Fc fragment, transferrinand fragments thereof, the C-terminal peptide of human chorionicgonadotropin, solvated random chains with large hydrodynamic volumeknown as XTEN, homo-amino acid repeats (HAP), proline-alanine-serinerepeats (PAS), albumin, afamin, alpha-fetoprotein, Vitamin D bindingprotein, polypeptides capable of binding under physiological conditionsto albumin or immunoglobulin constant regions, and combinations thereof.

[33] The glycoprotein of item [30], wherein said half-life-extendingmoiety conjugated to the glycoprotein is selected from the groupconsisting of hydroxyethyl starch (HES), polyethylene glycol (PEG),polysialic acids (PSAs), elastin-like polypeptides, heparosan polymers,hyaluronic acid and albumin binding ligands, e.g. fatty acid chains.

[34] The glycoprotein of any one of items [20] to [33], wherein theglycoprotein is a dimer.

[35] The dimeric glycoprotein of item [34], wherein the affinity of saiddimeric glycoprotein to the FVIII is greater than the affinity of amonomeric glycoprotein to said FVIII, wherein said monomericglycoprotein has the same amino acid sequence as the dimericglycoprotein.

[36] The glycoprotein of any one of items [20] to [35], wherein saidtruncated VWF has one or more amino acid substitution(s) relative to theamino acid sequence shown in SEQ ID NO:9, wherein the truncated VWFhaving said one or more amino acid substitutions has a greater affinityto FVIII than a truncated VWF consisting of the same amino acid sequenceexcept for said one or more amino acid substitutions relative to SEQ IDNO:9.

[37] The glycoprotein of item [36], wherein the affinity of saidglycoprotein to the FVIII is greater than the affinity of a referencepolypeptide, wherein the amino acid sequence of said referencepolypeptide is identical to the amino acid sequence of said glycoproteinexcept that the amino acid sequence of the truncated VWF of thereference polypeptide does not have said one or more substitutionsrelative to the amino acids sequence shown in SEQ ID NO:9.

[38] A composition comprising a population of glycoproteins as definedin any one of items [20] to [37], wherein the ratio of dimericglycoprotein to monomeric glycoprotein in the composition is greaterthan 1.0, preferably greater than 1.5, more preferably greater than 2.0,most preferably greater than 2.5.

[39] A pharmaceutical composition comprising a glycoprotein of any oneof items [20] to [37] and a pharmaceutically acceptable excipient.

[40] The pharmaceutical composition of item [39], wherein at least 50%,at least 60%, at least 70%, at least 80% or at least 90% of theglycoproteins in the composition are present as dimers.

[41] A glycoprotein as defined in any one of items [20] to [37] for usein the treatment of a blood coagulation disorder, said treatmentcomprising administering to a subject an effective amount of saidglycoprotein.

[42] The glycoprotein for use according to item [41], wherein saidtreatment further comprises administering to the subject an effectiveamount of a FVIII.

[43] The glycoprotein for use according to item [42], wherein the plasmaMRT of the FVIII is increased, and/or the clearance of the FVIII isreduced, by the co-administration of the glycoprotein, as compared to atreatment with the FVIII alone.

[44] The glycoprotein for use according to item [42] or [43], whereinthe frequency of administration of the FVIII is reduced as compared to atreatment with the FVIII alone.

[45] The glycoprotein for use according to any one of items [42] to[44], wherein the glycoprotein and/or the FVIII is/are administeredintravenously.

[46] The glycoprotein for use according to any one of items [42] to[44], wherein the glycoprotein and/or the FVIII is/are administeredsubcutaneously.

[47] The glycoprotein for use according to any one of items [42] to[46], wherein the glycoprotein and the FVIII are administeredseparately.

[48] The use of a glycoprotein as defined in any one of items [20] to[37] for increasing the plasma MRT of Factor VIII.

[49] The use of a glycoprotein as defined in any one of items [20] to[37] for reducing the clearance of administered FVIII from thecirculation.

[50] The use of item [48] or [49], wherein said Factor VIII isexogenously administered to a subject having hemophilia A.

[51] A pharmaceutical kit comprising (i) a FVIII and (ii) a glycoproteinas defined in any one of items [20] to [37] for simultaneous, separateor sequential use in the treatment of a blood coagulation disorder.

[52] A method of treating a blood coagulation disorder, comprisingadministering to a patient in need thereof an effective amount of aglycoprotein as defined in any one of items [20] to [37].

[53] A method of extending the circulatory half-life of an exogenouslyadministered FVIII, which comprises co-administering the glycoprotein ofany one of items [20] to [37].

DESCRIPTION OF THE DRAWINGS

FIG. 1: Mean residence time and clearance (mean) of D′D3-FP dimerquantified as albumin in rats, as determined in Example 8.1.

FIG. 2: Mean residence time and clearance (mean) of rVIII-SC quantifiedas FVIII antigen in rats, as determined in Example 8.1.

FIG. 3: Mean residence time and clearance (mean) of D′D3-FP dimerquantified as albumin in rats, as determined in Example 8.2.

FIG. 4: Mean residence time and clearance (mean) of full length FactorVIII quantified as FVIII antigen in rats, as determined in Example 8.2.

FIG. 5: Legend to the glycostructures shown in FIGS. 6 to 25

FIG. 6: Profil of lot B-140526 showing the neutral N-glycans

FIG. 7: Profil of lot B-140526 showing the mono-sialo N-glycans

FIG. 8: Profil of lot B-140526 showing the di-sialo N-glycans

FIG. 9: Profil of lot B-140526 showing the tri-sialo N-glycans

FIG. 10: Profil of lot B-140526 showing the tetra-sialo N-glycans

FIG. 11: Profil of lot B-140616KS showing the neutral N-glycans

FIG. 12: Profil of lot B-140616KS showing the mono-sialo N-glycans

FIG. 13: Profil of lot B-140616KS showing the di-sialo N-glycans

FIG. 14: Profil of lot B-140616KS showing the tri-sialo N-glycans

FIG. 15: Profil of lot B-140616KS showing the tetra-sialo N-glycans

FIG. 16: Profil of lot B-140825 showing the neutral N-glycans

FIG. 17: Profil of lot B-140825 showing the mono-sialo N-glycans

FIG. 18: Profil of lot B-140825 showing the di-sialo N-glycans

FIG. 19: Profil of lot B-140825 showing the tri-sialo N-glycans

FIG. 20: Profil of lot B-140825 showing the tetra-sialo N-glycans

FIG. 21: Profil of lot B-140623KS showing the neutral N-glycans

FIG. 22: Profil of lot B-140623KS showing the mono-sialo N-glycans

FIG. 23: Profil of lot B-140623KS showing the di-sialo N-glycans

FIG. 24: Profil of lot B-140623KS showing the tri-sialo N-glycans

FIG. 25: Profil of lot B-140623KS showing the tetra-sialo N-glycans

FIG. 26: Quantitative determination of N-glycans with two or moreterminal and non-sialylated galactose residues of the comparative sampleB140526. The first column shows the quantitative distribution of allN-glycans for neutral, mono-sialo, di-sialo, tri-sialo and tetra-sialoN-glycans adding up to 100%. The second column shows the percentage(relating to the 100% of all N-glycans) of N-glycans with two or moreterminal and non-sialylated galactose residues. In the present sampleonly neutral, mono-sialo and di-sialo N-glycans having two or moreterminal and non-sialylated galactose residues were detected. The thirdcolumn shows the percentage (relating to the 100% of all N-glycans) ofN-glycans with three or more terminal and non-sialylated galactoseresidues. In the present sample only neutral N-glycans having three ormore terminal and non-sialylated galactose residues were detected.

FIG. 27: Quantitative determination of N-glycans with two or moreterminal and non-sialylated galactose residues of sample B140616KSaccording to the invention. The first column shows the quantitativedistribution of all N-glycans for neutral, mono-sialo, di-sialo,tri-sialo and tetra-sialo N-glycans adding up to 100%. The second columnshows the percentage (relating to the 100% of all N-glycans) ofN-glycans with two or more terminal and non-sialylated galactoseresidues. In the present sample only neutral, mono-sialo and di-sialoN-glycans having two or more terminal and non-sialylated galactoseresidues were detected. The third column shows the percentage (relatingto the 100% of all N-glycans) of N-glycans with three or more terminaland non-sialylated galactose residues. In the present sample onlyneutral N-glycans having three or more terminal and non-sialylatedgalactose residues were detected.

FIG. 28: Quantitative determination of N-glycans with two or moreterminal and non-sialylated galactose residues of sample B140825according to the invention. The first column shows the quantitativedistribution of all N-glycans for neutral, mono-sialo, di-sialo,tri-sialo and tetra-sialo N-glycans adding up to 100%. The second columnshows the percentage (relating to the 100% of all N-glycans) ofN-glycans with two or more terminal and non-sialylated galactoseresidues. In the present sample only neutral, mono-sialo and di-sialoN-glycans having two or more terminal and non-sialylated galactoseresidues were detected. The third column shows the percentage (relatingto the 100% of all N-glycans) of N-glycans with three or more terminaland non-sialylated galactose residues. In the present sample onlyneutral N-glycans having three or more terminal and non-sialylatedgalactose residues were detected.

FIG. 29: Quantitative determination of N-glycans with two or moreterminal and non-sialylated galactose residues of sample B140623KSaccording to the invention. The first column shows the quantitativedistribution of all N-glycans for neutral, mono-sialo, di-sialo,tri-sialo and tetra-sialo N-glycans adding up to 100%. The second columnshows the percentage (relating to the 100% of all N-glycans) ofN-glycans with two or more terminal and non-sialylated galactoseresidues. In the present sample only neutral, mono-sialo and di-sialoN-glycans having two or more terminal and non-sialylated galactoseresidues were detected. The third column shows the percentage (relatingto the 100% of all N-glycans) of N-glycans with three or more terminaland non-sialylated galactose residues. In the present sample onlyneutral N-glycans having three or more terminal and non-sialylatedgalactose residues were detected.

DETAILED DESCRIPTION

In a first aspect, the present invention pertains to a method ofproducing a glycoprotein comprising sialylated N-glycans, whichcomprises (i) providing cells comprising a nucleic acid encoding apolypeptide comprising a truncated von Willebrand Factor (VWF), and (ii)culturing said cells at a temperature of less than 36.0° C. Preferably,the N-glycans of the produced glycoprotein have an increasedsialylation, and/or a high degree of sialylation.

In a second aspect, the present invention pertains to a method ofproducing a glycoprotein comprising sialylated N-glycans, whichcomprises (i) providing cells comprising a nucleic acid encoding apolypeptide comprising a truncated von Willebrand Factor (VWF) and arecombinant nucleic acid encoding an α-2,3-sialyltransferase and/or anα-2,6-sialyltransferase, and (ii) culturing the cells under conditionsthat allow expression of the glycoprotein and of thesialyltransferase(s). Preferably, the N-glycans of the producedglycoprotein have an increased sialylation, and/or a high sialylation.

The term “glycoprotein”, as used herein, refers to a protein orpolypeptide that comprises one or more covalently linked oligosaccharidechains. The oligosaccharide chains may be composed of a singleunbranched chain of sugar residues or may be composed of a chain ofsugar residues that branches one or more times.

“N-linked glycans” are oligosaccharides that are covalently linked toasparagine residues of a polypeptide. Terminal galactoses on suchN-linked glycans may be modified by the attachment of an α-2,3- or anα-2,6-linked sialic acid (as shown in FIGS. 5 to 25). Preferably theterminal galactoses are D-galactoses. N-glycans are usually branched andcan be, for example, of a bi-, tri- or tetra-antennary type, so thatthere could be two, three or four terminal galactose residues in oneN-glycan, which may be sialylated to varying degrees or be allnon-sialylated. “Terminal” refers to the most distant position in agiven branch of an N-glycan from the attachment point of the N-glycan tothe peptidic chain of the glycoprotein of the invention.

The term “sialic acid” refers to the N- or O-substituted derivatives ofneuraminic acid usually found as terminal monosaccharides of animaloligosaccharides (for review, see Varkis (1992) Glycobiology vol. 2 no.1 pp. 25-40). The most common sialic acid is N-acetyl neuraminic acid.An “increased sialylation” means that at least 75% of the N-glycans ofthe glycoprotein comprise, on average, at least one sialic acid moiety.By way of non-limiting example an “increased sialylation of at least75%” is determined as in Example 6 of the present invention, i.e. byenzymatically cleaving all N-glycans from a given glycoprotein ofinterest and then determining the amount of cleaved N-glycans with nosialic acids (“asialo N-glycans”) and the total amount of all cleavedN-glycans. A “sialylation of at least 75%” corresponds then to an amountof 25% of asialo N-glycans or less of the total amount of all cleavedN-glycans.

Increased sialylation is of importance for maintaining a giventherapeutic glycoprotein longer in the circulation since glycoproteinswith a reduced sialylation bind to the asialoglycoprotein receptor(ASGP-R) and are then—after receptor mediated endocytosis—finallydegraded.

The ASGP-R is expressed exclusively by parenchymal hepatocytes, whichcontain 100,000-500,000 binding sites per cell. These receptors arerandomly distributed over the sinusoidal plasma membrane facing theblood capillaries. Their main function is to maintain plasmaglycoprotein homeostasis by recognition, binding and endocytosis ofasialoglycoproteins (Stokmaier et al (2009) Bioorganic & MedicinalChemistry, 7254-7264).

The human ASGP-R consists of two homologous subunits, designated H1 andH2, which form a non-covalent heteroligomeric complex with an estimatedratio of 2-5:1, respectively. This ASGP-R complex binds to glycoproteinsexposing glycostructures with non-sialylated terminal D-galactose andN-acetyl-D-galactoseamin residues. It has been found that the bindingaffinity of glycostructures to the ASGP-R strongly depends on thevalency of the ligand. Whereas the affinity of a single D-galactoseresidue is only in the millimolar range, bi-, tri- and tetraantennarydesialylated glycans bind with dissociation constants of 10⁻⁶, 5×10⁻⁹and 10⁻⁹ M, respectively.

Therefore in particular preferred embodiments of the invention theglycoprotein of the invention which is characterized by a high degree ofsialylation of its N-glycans has a particular low amount of N-glycanswith multivalent terminal and non-sialylated galactose residues,including a particular low amount of N-glycans with two or more terminaland non-sialylated galactose residues, and even more preferred aparticular low amount of N-glycans with three or more terminal andnon-sialylated galactose residues.

In a first step, the methods of the invention comprise the step ofproviding cells comprising a nucleic acid encoding a polypeptidecomprising a truncated von Willebrand Factor (VWF).

The Truncated VWF

The term “von Willebrand Factor” (VWF) as used herein includes naturallyoccurring (native) VWF, but also variants thereof retaining at least theFVIII binding activity of naturally occurring VWF, e.g. sequencevariants where one or more residues have been inserted, deleted orsubstituted. The FVIII binding activity is determined by a FVIII-VWFbinding assay as described in Example 11.

The preferred VWF in accordance with this invention is human VWFrepresented by the amino acid sequence shown in SEQ ID NO:9. The cDNAencoding SEQ ID NO:9 is shown in SEQ ID NO:8.

The gene encoding human native VWF is transcribed into a 9 kb mRNA whichis translated into a pre-propolypeptide of 2813 amino acids with anestimated molecular weight of 310,000 Da. The pre-propolypeptidecontains an N-terminal 22 amino acids signal peptide, followed by a 741amino acid pro-polypeptide (amino acids 23-763 of SEQ ID NO:9) and themature subunit (amino acids 764-2813 of SEQ ID NO:9). Cleavage of the741 amino acids propolypeptide from the N-terminus results in mature VWFconsisting of 2050 amino acids. The amino acid sequence of the humannative VWF pre-propolypeptide is shown in SEQ ID NO:9. Unless indicatedotherwise, the amino acid numbering of VWF residues in this applicationrefers to SEQ ID NO:9, even if the VWF molecule does not comprise allresidues of SEQ ID NO:9.

The propolypeptide of native VWF comprises multiple domains. Differentdomain annotations can be found in the literature (see, e.g. Zhou et al.(2012) Blood 120(2): 449-458). The following domain annotation of nativepre-propolypeptide of VWF is applied in this application:

D1-D2-D′-D3-A1-A2-A3-D4-C1-C2-C3-C4-05-C6-CK

With reference to SEQ ID NO:9, the D′ domain consists of amino acids764-865; and the D3 domain consists of amino acids 866-1242.

The feature “truncated” means that the polypeptide does not comprise theentire amino acid sequence of mature VWF (amino acids 764-2813 of SEQ IDNO:9). Typically, the truncated VWF does not comprise all amino acids764-2813 of SEQ ID NO:9 but only a fragment thereof. A truncated VWF mayalso be referred to as a VWF fragment, or in the plural as VWFfragments.

Typically, the truncated VWF is capable of binding to a Factor VIII.Preferably, the truncated VWF is capable of binding to the mature formof human native Factor VIII. In another embodiment, the truncated VWF iscapable of binding to the single-chain Factor VIII consisting of theamino acid sequence SEQ ID NO:10. Binding of the truncated VWF to FactorVIII can be determined by a FVIII-VWF binding assay as described inExample 11.

The truncated VWF of the present invention preferably comprises orconsists of an amino acid sequence having a sequence identity of atleast 90% to amino acids 776 to 805 of SEQ ID NO:9 and is capable ofbinding to FVIII. In preferred embodiments the truncated VWF comprisesor consists of an amino acid sequence having a sequence identity of atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%, toamino acids 776 to 805 of SEQ ID NO:9 and is capable of binding toFVIII. Most preferably, the truncated VWF comprises or consists of aminoacids 776 to 805 of SEQ ID NO:9. Unless indicated otherwise herein,sequence identities are determined over the entire length of thereference sequence (e.g. amino acids 776 to 805 of SEQ ID NO:9).

The truncated VWF of the present invention preferably comprises orconsists of an amino acid sequence having a sequence identity of atleast 90% to amino acids 766 to 864 of SEQ ID NO:9 and is capable ofbinding to FVIII. In preferred embodiments the truncated VWF comprisesor consists of an amino acid sequence having a sequence identity of atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%, toamino acids 766 to 864 of SEQ ID NO:9 and is capable of binding toFVIII. Most preferably, the truncated VWF comprises or consists of aminoacids 766 to 864 of SEQ ID NO:9.

In another preferred embodiment, the truncated VWF consists of (a) anamino acid sequence having a sequence identity of at least 90% to aminoacids 764 to 1242 of SEQ ID NO:9, or (b) a fragment thereof, providedthat the truncated VWF is still capable of binding to FVIII. Morepreferably, the truncated VWF consists of (a) an amino acid sequencehaving a sequence identity of at least 95%, at least 96%, at least 97%,at least 98%, or at least 99%, to amino acids 764 to 1242 of SEQ IDNO:9, or (b) a fragment thereof, provided that the truncated VWF isstill capable of binding to FVIII. Most preferably, the truncated VWFconsists of (a) amino acids 764 to 1242 of SEQ ID NO:9, or (b) afragment thereof, provided that the truncated VWF is still capable ofbinding to FVIII.

As described in more detail below, the method of the invention comprisesproviding cells comprising a nucleic acid encoding the polypeptidecomprising the truncated VWF. The nucleic acid is introduced intosuitable host cells by techniques that are known per se.

In a preferred embodiment, the nucleic acid in the host cell encodes (a)an amino acid sequence having a sequence identity of at least 90% toamino acids 1 to 1242 of SEQ ID NO:9, or (b) a fragment thereof,provided that the truncated mature VWF is still capable of binding toFVIII. More preferably, the nucleic acid encodes (a) an amino acidsequence having a sequence identity of at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, to amino acids 1 to 1242 ofSEQ ID NO:9, or (b) a fragment thereof, provided that the truncated VWFis still capable of binding to FVIII. Most preferably, the nucleic acidencodes (a) amino acids 1 to 1242 of SEQ ID NO:9, or (b) a fragmentthereof, provided that the truncated VWF is still capable of binding toFVIII. Especially if the glycoprotein eventually produced is a dimer,the nucleic acid will comprise a sequence encoding amino acids 1 to 763of VWF (e.g. SEQ ID NO:9), even if the truncated VWF in the glycoproteindoes not comprise amino acids 1 to 763 of VWF (e.g. SEQ ID NO:9).

In other embodiments the truncated VWF comprises or consists of one ofthe following amino acid sequences, each referring to SEQ ID NO:9:776-805; 766-805; 764-805; 776-810; 766-810; 764-810; 776-815; 766-815;764-815; 776-820; 766-820; 764-820; 776-825; 766-825; 764-825; 776-830;766-830; 764-830; 776-835; 766-835; 764-835; 776-840; 766-840; 764-840;776-845; 766-845; 764-845; 776-850; 766-850; 764-850; 776-855; 766-855;764-855; 776-860; 766-860; 764-860; 776-864; 766-864; 764-864; 776-865;766-865; 764-865; 776-870; 766-870; 764-870; 776-875; 766-875; 764-875;776-880; 766-880; 764-880; 776-885; 766-885; 764-885; 776-890; 766-890;764-890; 776-895; 766-895; 764-895; 776-900; 766-900; 764-900; 776-905;766-905; 764-905; 776-910; 766-910; 764-910; 776-915; 766-915; 764-915;776-920; 766-920; 764-920; 776-925; 766-925; 764-925; 776-930; 766-930;764-930; 776-935; 766-935; 764-935; 776-940; 766-940; 764-940; 776-945;766-945; 764-945; 776-950; 766-950; 764-950; 776-955; 766-955; 764-955;776-960; 766-960; 764-960; 776-965; 766-965; 764-965; 776-970; 766-970;764-970; 776-975; 766-975; 764-975; 776-980; 766-980; 764-980; 776-985;766-985; 764-985; 776-990; 766-990; 764-990; 776-995; 766-995; 764-995;776-1000; 766-1000; 764-1000; 776-1005; 766-1005; 764-1005; 776-1010;766-1010; 764-1010; 776-1015; 766-1015; 764-1015; 776-1020; 766-1020;764-1020; 776-1025; 766-1025; 764-1025; 776-1030; 766-1030; 764-1030;776-1035; 766-1035; 764-1035; 776-1040; 766-1040; 764-1040; 776-1045;766-1045; 764-1045; 776-1050; 766-1050; 764-1050; 776-1055; 766-1055;764-1055; 776-1060; 766-1060; 764-1060; 776-1065; 766-1065; 764-1065;776-1070; 766-1070; 764-1070; 776-1075; 766-1075; 764-1075; 776-1080;766-1080; 764-1080; 776-1085; 766-1085; 764-1085; 776-1090; 766-1090;764-1090; 776-1095; 766-1095; 764-1095; 776-1100; 766-1100; 764-1100;776-1105; 766-1105; 764-1105; 776-1110; 766-1110; 764-1110; 776-1115;766-1115; 764-1115; 776-1120; 766-1120; 764-1120; 776-1125; 766-1125;764-1125; 776-1130; 766-1130; 764-1130; 776-1135; 766-1135; 764-1135;776-1140; 766-1140; 764-1140; 776-1145; 766-1145; 764-1145; 776-1150;766-1150; 764-1150; 776-1155; 766-1155; 764-1155; 776-1160; 766-1160;764-1160; 776-1165; 766-1165; 764-1165; 776-1170; 766-1170; 764-1170;776-1175; 766-1175; 764-1175; 776-1180; 766-1180; 764-1180; 776-1185;766-1185; 764-1185; 776-1190; 766-1190; 764-1190; 776-1195; 766-1195;764-1195; 776-1200; 766-1200; 764-1200; 776-1205; 766-1205; 764-1205;776-1210; 766-1210; 764-1210; 776-1215; 766-1215; 764-1215; 776-1220;766-1220; 764-1220; 776-1225; 766-1225; 764-1225; 776-1230; 766-1230;764-1230; 776-1235; 766-1235; 764-1235; 776-1240; 766-1240; 764-1240;776-1242; 766-1242; 764-1242; 764-1464; 764-1250; 764-1041; 764-828;764-865; 764-1045; 764-1035; 764-1128; 764-1198; 764-1268; 764-1261;764-1264; 764-1459; 764-1463; 764-1464; 764-1683; 764-1873; 764-1482;764-1479; 764-1672; and 764-1874.

In certain embodiments the truncated VWF has an internal deletionrelative to mature wild type VWF. For example, the A1, A2, A3, D4, C1,C2, C3, C4, C5, C6 domains or combinations thereof may be deleted, andthe D′ domain, the D3 domain and the CK domain is retained. In furtherembodiments the truncated VWF does not comprise the binding sites forplatelet glycoprotein Ibα (GPlbα), collagen and/or integrin αllbβlll(RGDS sequence within the C1 domain). In other embodiments, thetruncated VWF does not comprise the cleavage site (Tyr1605-Met1606) forADAMTS13 which is located at the central A2 domain of VWF. In yetanother embodiment, the truncated VWF does not comprise the bindingsites for GPlbα, and/or does not comprise the binding site for collagen,and/or does not comprise the binding site for integrin αllbβlll, and/orit does not comprise the cleavage site (Tyr1605-Met1606) for ADAMTS13which is located at the central A2 domain of VWF.

In other embodiments the truncated VWF comprises or consists of an aminoacid sequence that has a sequence identity of at least 90%, or at least91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%,or at least 96%, or at least 97%, or at least 98%, or at least 99%, toone of the amino acid sequences recited in the preceding paragraph,provided that the truncated VWF is capable of binding to FVIII.

A glycoprotein is termed a “dimer” in the present invention if twomonomers of the glycoprotein are linked covalently. Preferably the twomonomeric subunits are covalently linked via at least one disulfidebridge, e.g. by one, two, three or four disulfide bridges. The cysteineresidues forming the at least one disulfide bridge are preferablylocated within the truncated VWF portion of the glycoprotein. In oneembodiment, these cysteine residues are Cys-1142, Cys-1222, Cys-1225,Cys-1227 and combinations thereof.

If the glycoprotein of the invention is a dimer, the truncated VWFpreferably comprises or consists of two polypeptides each with an aminoacid sequence having a sequence identity of at least 90% to amino acids764 to 1099, amino acids 764 to 1142, amino acids 764 to 1222, aminoacids 764 to 1225, or amino acids 764 to 1227 of SEQ ID NO:9 and iscapable of binding to FVIII. In preferred embodiments the truncated VWFcomprises or consists of an amino acid sequence having a sequenceidentity of at least 95%, at least 96%, at least 97%, at least 98%, orat least 99%, to amino acids 764 to 1099, amino acids 764 to 1142, aminoacids 764 to 1222, amino acids 764 to 1225, or amino acids 764 to 1227of SEQ ID NO:9 and is capable of binding to FVIII. Most preferably, thetruncated VWF comprises or consists of amino acids 764 to 1099, aminoacids 764 to 1142, amino acids 764 to 1222, amino acids 764 to 1225, oramino acids 764 to 1227 of SEQ ID NO:9.

The truncated VWF may be any one of the VWF fragments disclosed in WO2013/106787 A1, WO 2014/198699 A2, WO 2011/060242 A2 or WO 2013/093760A2, the disclosure of which is incorporated herein by reference.

Further Components of the Polypeptide

In addition to the truncated VWF, the glycoprotein may further comprisesa half-life extending moiety. The half-life-extending moiety may be aheterologous amino acid sequence fused to the truncated VWF.Alternatively, the half-life-extending moiety may be chemicallyconjugated to the polypeptide comprising the truncated VWF by a covalentbond different from a peptide bond.

In certain embodiments of the invention, the half-life of theglycoprotein is extended by chemical modification, e.g. attachment of ahalf-life extending moiety such as polyethylene glycol (PEGylation),glycosylated PEG, hydroxyl ethyl starch (HESylation), polysialic acids,elastin-like polypeptides, heparosan polymers or hyaluronic acid. Inanother embodiment, the glycoprotein is conjugated to a HLEP such asalbumin via a chemical linker. The principle of this conjugationtechnology has been described in an exemplary manner by Conjuchem LLC(see, e.g., U.S. Pat. No. 7,256,253).

In other embodiments, the half-life-extending moiety is a half-lifeenhancing protein (HLEP). Preferably, the HLEP is an albumin or afragment thereof. The N-terminus of the albumin may be fused to theC-terminus of the truncated VWF. Alternatively, the C-terminus of thealbumin may be fused to the N-terminus of the truncated VWF. One or moreHLEPs may be fused to the N- or C-terminal part of VWF provided thatthey do not to interfere with or abolish the binding capability of thetruncated VWF to FVIII.

In one embodiment the polypeptide has the following structure:

tVWF−L1−H,   [formula 1]

Wherein tVWF is the truncated VWF, L1 is a chemical bond or a linkersequence, and H is a HLEP.

L1 may be a chemical bond or a linker sequence consisting of one or moreamino acids, e.g. of 1 to 50, 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 5or 1 to 3 (e.g. 1 , 2 or 3) amino acids and which may be equal ordifferent from each other. Usually, the linker sequences are not presentat the corresponding position in the wild-type VWF. Examples of suitableamino acids present in L1 include Gly and Ser. The linker should benon-immunogenic and may be a non-cleavable or cleavable linker.Non-cleavable linkers may be comprised of alternating glycine and serineresidues as exemplified in WO2007/090584. In another embodiment of theinvention the peptidic linker between the truncated VWF moiety and thealbumin moiety consists of peptide sequences, which serve as naturalinterdomain linkers in human proteins. Preferably such peptide sequencesin their natural environment are located close to the protein surfaceand are accessible to the immune system so that one can assume a naturaltolerance against this sequence. Examples are given in WO2007/090584.Cleavable linker sequences are described, e.g., in WO 2013/120939 A1.

Preferred HLEP sequences are described infra. Likewise encompassed bythe invention are fusions to the exact “N-terminal amino acid” of therespective HLEP, or fusions to the “N-terminal part” of the respectiveHLEP, which includes N-terminal deletions of one or more amino acids ofthe HLEP. The polypeptide may comprise more than one HLEP sequence, e.g.two or three HLEP sequences. These multiple HLEP sequences may be fusedto the C-terminal part of VWF in tandem, e.g. as successive repeats.

In another embodiment of the invention, the half-life of the complex ofthe invention is extended by chemical modification, e.g. attachment of ahalf-life extending moiety such as polyethylene glycol (PEGylation),glycosylated PEG, hydroxyl ethyl starch (HESylation), polysialic acids,elastin-like polypeptides, heparosan polymers or hyaluronic acid. Inanother embodiment, the glycoprotein of the invention is conjugated to aHLEP such as albumin via a chemical linker. The principle of thisconjugation technology has been described in an exemplary manner byConjuchem LLC (see, e.g., U.S. Pat. No. 7,256,253).

Half-Life Enhancing Polypeptides (HLEPs)

Preferably, the half-life extending moiety is a half-life extendingpolypeptide (HLEP), more preferably HLEP is selected from albumin orfragments thereof, immunoglobulin constant region and portions thereof,e.g. the Fc fragment, solvated random chains with large hydrodynamicvolume (e.g. XTEN (Schellenberger et al. 2009; Nature Biotechnol.27:1186-1190), homo-amino acid repeats (HAP) or proline-alanine-serinerepeats (PAS)), afamin, alpha-fetoprotein, Vitamin D binding protein,transferrin or variants thereof, carboxyl-terminal peptide (CTP) ofhuman chorionic gonadotropin-f3 subunit, polypeptides or lipids capableof binding under physiological conditions to albumin or immunoglobulinconstant region.

A “half-life enhancing polypeptide” as used herein is preferablyselected from the group consisting of albumin, a member of thealbumin-family, the constant region of immunoglobulin G and fragmentsthereof, region and polypeptides capable of binding under physiologicalconditions to albumin, to members of the albumin family as well as toportions of an immunoglobulin constant region. It may be a full-lengthhalf-life-enhancing protein described herein (e.g. albumin, a member ofthe albumin-family or the constant region of immunoglobulin G) or one ormore fragments thereof that are capable of stabilizing or prolonging thetherapeutic activity or the biological activity of the coagulationfactor. Such fragments may be of 10 or more amino acids in length or mayinclude at least about 15, at least about 20, at least about 25, atleast about 30, at least about 50, at least about 100, or morecontiguous amino acids from the HLEP sequence or may include part or allof specific domains of the respective HLEP, as long as the HLEP fragmentprovides a functional half-life extension of at least 25% compared tothe respective polypeptide without the HLEP.

The HLEP portion of the glycoprotein may be a variant of a wild typeHLEP. The term “variants” includes insertions, deletions andsubstitutions, either conservative or non-conservative, where suchchanges do not substantially alter the FVIII-binding activity of thetruncated VWF.

In particular, the proposed truncated VWF HLEP fusion constructs of theinvention may include naturally occurring polymorphic variants of HLEPsand fragments of HLEPs. The HLEP may be derived from any vertebrate,especially any mammal, for example human, monkey, cow, sheep, or pig.Non-mammalian HLEPs include, but are not limited to, hen and salmon.

Albumin as HLEP

The terms, “human serum albumin” (HSA) and “human albumin” (HA) and“albumin” (ALB) are used interchangeably in this application. The terms“albumin” and “serum albumin” are broader, and encompass human serumalbumin (and fragments and variants thereof) as well as albumin fromother species (and fragments and variants thereof).

As used herein, “albumin” refers collectively to albumin polypeptide oramino acid sequence, or an albumin fragment or variant, having one ormore functional activities (e.g., biological activities) of albumin. Inparticular, “albumin” refers to human albumin or fragments thereof,especially the mature form of human albumin as shown in SEQ ID NO:11herein or albumin from other vertebrates or fragments thereof, oranalogs or variants of these molecules or fragments thereof.

In particular, the proposed truncated VWF fusion constructs of theinvention may include naturally occurring polymorphic variants of humanalbumin and fragments of human albumin. Generally speaking, an albuminfragment or variant will be at least 10, preferably at least 40, mostpreferably more than 70 amino acids long.

Preferred embodiments of the invention include albumin variants withenhanced binding to the FcRn receptor. Such albumin variants may lead toa longer plasma half-life of a truncated VWF albumin variant fusionprotein as compared to a truncated VWF fusion with a wild-type albumin.

The albumin portion of the proposed VWF fusion constructs of theinvention may comprise at least one subdomain or domain of HA orconservative modifications thereof.

Immunoglobulins as HLEPs

Immunoglobulin G (IgG) constant regions (Fc) are known in the art toincrease the half-life of therapeutic proteins (Dumont J A et al. 2006.BioDrugs 20:151-160). The IgG constant region of the heavy chainconsists of 3 domains (CH1-CH3) and a hinge region. The immunoglobulinsequence may be derived from any mammal, or from subclasses IgG1, IgG2,IgG3 or IgG4, respectively. IgG and IgG fragments without anantigen-binding domain may also be used as HLEPs. The therapeuticpolypeptide portion is connected to the IgG or the IgG fragmentspreferably via the hinge region of the antibody or a peptidic linker,which may even be cleavable. Several patents and patent applicationsdescribe the fusion of therapeutic proteins to immunoglobulin constantregions to enhance the therapeutic protein's in vivo half-lives. US2004/0087778 and WO 2005/001025 describe fusion proteins of Fc domainsor at least portions of immunoglobulin constant regions withbiologically active peptides that increase the half-life of the peptide,which otherwise would be quickly eliminated in vivo. Fc-IFN-β fusionproteins were described that achieved enhanced biological activity,prolonged circulating half-life and greater solubility (WO 2006/000448).Fc-EPO proteins with a prolonged serum half-life and increased in vivopotency were disclosed (WO 2005/063808) as well as Fc fusions with G-CSF(WO 2003/076567), glucagon-like peptide-1 (WO 2005/000892), clottingfactors (WO 2004/101740) and interleukin-10 (U.S. Pat. No. 6,403,077),all with half-life enhancing properties.

Various HLEPs which can be used in accordance with this invention aredescribed in detail in WO 2013/120939 A1.

Nucleic Acid

The nucleic acid encoding the polypeptide to be expressed can beprepared according to methods known in the art. Based on the cDNAsequence of VWF (SEQ ID NO:8), recombinant DNA encoding theabove-mentioned truncated VWF constructs can be designed and generated.

Even if the glycoprotein which is secreted by the host cells does notcomprise amino acids 1 to 763 of VWF, it is preferred that the nucleicacid (e.g. the DNA) encoding the intracellular precursor of theglycoprotein comprises a nucleotide sequence encoding an amino acidsequence having a sequence identity of at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, to amino acids 23 to 763 orpreferably to amino acids 1 to 763 of SEQ ID NO:9. Most preferably, thenucleic acid (e.g. the DNA) encoding the intracellular precursor of theglycoprotein comprises a nucleotide sequence encoding amino acids 23 to763 of SEQ ID NO:9, or amino acids 1 to 763 of SEQ ID NO:9.

Constructs in which the DNA contains the entire open reading frameinserted in the correct orientation into an expression plasmid may beused for protein expression. Typical expression vectors containpromoters that direct the synthesis of large amounts of mRNAcorresponding to the inserted nucleic acid in the plasmid-bearing cells.They may also include an origin of replication sequence allowing fortheir autonomous replication within the host organism, and sequencesthat increase the efficiency with which the synthesized mRNA istranslated. Stable long-term vectors may be maintained as freelyreplicating entities by using regulatory elements of, for example,viruses (e.g., the OriP sequences from the Epstein Barr Virus genome).Cell lines may also be produced that have integrated the vector into thegenomic DNA, and in this manner the gene product is produced on acontinuous basis.

Typically, the cells to be provided are obtained by introducing thenucleic acid encoding a polypeptide comprising the truncated VWF intomammalian host cells.

Host Cells

Any host cell susceptible to cell culture, and to expression ofglycoproteins, may be utilized in accordance with the present invention.In certain embodiments, a host cell is mammalian. Non-limiting examplesof mammalian cells that may be used in accordance with the presentinvention include BALB/c mouse myeloma line (NSO/1, ECACC No: 85110503);human retinoblasts (PER.C6 (CruCell, Leiden, The Netherlands)); monkeykidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol., 36:59, 1977); babyhamster kidney cells (BHK, ATCC CCL10); Chinese hamster ovary cells+/−DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216,1980); mouse sertoli cells (TM4, Mather, Biol. Reprod., 23:243 251,1980); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1 587); human cervical carcinoma cells(HeLa, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (HepG2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals NY. Acad. Sci.,383:44-68, 1982); MRC 5 cells; PS4 cells; human amniocyte cells (CAP);and a human hepatoma line (Hep G2). Preferably, the cell line is arodent cell line, especially a hamster cell line such as CHO or BHK.

Methods suitable for introducing nucleic acids sufficient to achieveexpression of a glycoprotein of interest into mammalian host cells areknown in the art. See, for example, Gething et al., Nature, 293:620-625,1981; Mantei et al., Nature, 281:40-46, 1979; Levinson et al. EP117,060; and EP 117,058. For mammalian cells, common methods ofintroducing genetic material into mammalian cells include the calciumphosphate precipitation method of Graham and van der Erb (Virology,52:456-457, 1978) or the Iipofectamine™ (Gibco BRL) Method ofHawley-Nelson (Focus 15:73, 1993). General aspects of mammalian cellhost system transformations have been described by Axel in U.S. Pat. No.4,399,216. For various techniques for introducing genetic material intomammalian cells, see Keown et al., Methods in Enzymology, 1989, Keown etal., Methods in Enzymology, 185:527-537, 1990, and Mansour et al.,Nature, 336:348-352, 1988.

Culturing the Cells

In a second step, the method of the first aspect of the inventioncomprises culturing the cells at a temperature of less than 36.0° C. Inthe method of the second aspect, the method comprises culturing thecells under conditions that allow expression of the glycoprotein.

The basal medium chosen for culturing the host cell line is not criticalto the present invention and may be any one of, or combination of, thoseknown to the art which are suitable for culturing mammalian cells. Mediasuch as Dulbecco's Modified Eagle Medium, Ham's F-12 Medium, Eagle'sMinimal Essential Medium and RPMI-1640 Medium and the like arecommercially available. The addition of growth factors such asrecombinant insulin is optional. In one embodiment, the medium is“protein-free” in the sense that it is either completely free of anyprotein or at least are free of any protein that is not recombinantlyproduced. Human serum albumin may be used as a serum-free culturesupplement for the production of the glycoprotein. Preferably, themedium contains a protease inhibitor, such as a serine proteaseinhibitor, which is suitable for tissue culture and which is ofsynthetic or vegetable origin.

Generally, the present invention may be used with any cell culturemethod that is amenable to the expression of glycoproteins. For example,cells may be grown in batch or fed-batch cultures, where the culture isterminated after sufficient expression of the glycoprotein, after whichthe expressed glycoprotein is harvested. Alternatively, cells may begrown in continuous cultures (e.g. perfusion cultures), where theculture is not terminated and new nutrients and other components areperiodically or continuously added to the culture, during which theexpressed glycoprotein is harvested periodically or continuously. Thelatter embodiment is preferred if the method comprises a temperatureshift as described hereinbelow. The culture can be any conventional typeof culture, such as batch, fed-batch or continuous, but is preferablycontinuous. Suitable continuous cultures include perfusion culture.

Cells may be grown in any convenient volume chosen by the practitioner.For example, cells may be grown in small scale reaction vessels rangingin volume from a few milliliters to several liters. Alternatively, cellsmay be grown in large scale commercial bioreactors ranging in volumefrom approximately at least 1 liter to 10, 100, 250, 500, 1000, 2500,5000, 8000, 10,000, 12,000 liters or more, or any volume in between. Theculture is typically carried out in a bioreactor, which is usually astainless steel, glass or plastic vessel of 1 (one) to 10000 (tenthousand) litres capacity, for example 5, 10, 50, 100, 1000, 2500, 5000or 8000 litres. The vessel is usually rigid but flexible plastic bagscan be used, particularly for smaller volumes. These are generally ofthe ‘single use’ type.

Mammalian cells such as CHO and BHK cells are generally cultured assuspension cultures. That is to say, the cells are suspended in themedium, rather than adhering to a solid support. The cells mayalternatively be immobilized on a carrier, in particular on amicrocarrier. Porous carriers, such as Cytoline®, Cytopore® or Cytodex®,may be particularly suitable.

To obtain a high sialylation, the cells (e.g. CHO cells) are preferablycultured at a decreased temperature, e.g. at less than 36.0° C.“Decreased temperature” refers to a temperature that is lower than theoptimum temperature or normal temperature for growth of the respectivecell line. For most mammalian cells the normal temperature is 37° C. Itis therefore preferred according to the invention that the cells (e.g.CHO cells) are cultured at a decreased temperature of 30.0 to 36.0° C.,30.5 to 35.5° C., 31.0 to 35.0° C., 31.5 to 34.5° C., 32.0 to 34.0° C.,or 32.5 to 33.5° C. Preferably, the cells are cultured at a decreasedtemperature of 30.0° C.±1.0° C., 31.0° C.±1.0° C., 32.0° C.±1.0° C.,33.0° C.±1.0° C., 34.0° C.±1.0° C., or 35.0° C.±1.0° C.

The decreased temperature is maintained for a time period that issufficient to increase the sialylation of the glycoprotein to beexpressed. Preferably, the decreased temperature is maintained for atleast 1 hour, at least 6 hours, at least 12 hours, at least 18 hours, atleast 24 hours, at least 48 hours, at least 72 hours, at least 96 hours,at least 120 hours, or at least 144 hours. In other embodiments, thedecreased temperature is maintained for 1 hour to 8 weeks, 6 hours to 6weeks, 12 hours to 5 weeks, 18 hours to 4 weeks, 24 hours to 3 weeks, 48hours to 14 days, 72 hours to 10 days, or 3 to 7 days.

To accomplish this, a culture may be subjected to one or moretemperature shifts during the course of the culture. When shifting thetemperature of a culture, the temperature shift may be relativelygradual. For example, it may take several hours or days to complete thetemperature change. Alternatively, the temperature shift may berelatively abrupt. The temperature may be steadily increased ordecreased during the culture process.

Alternatively, the temperature may be increased or decreased by discreteamounts at various times during the culture process. The subsequenttemperature(s) or temperature range(s) may be lower than or higher thanthe initial or previous temperature(s) or temperature range(s). One ofordinary skill in the art will understand that multiple discretetemperature shifts are encompassed in this embodiment. For example, thetemperature may be shifted once (either to a higher or lower temperatureor temperature range), the cells maintained at this temperature ortemperature range for a certain period of time, after which thetemperature may be shifted again to a new temperature or temperaturerange, which may be either higher or lower than the temperature ortemperature range of the previous temperature or temperature range. Thetemperature of the culture after each discrete shift may be constant ormay be maintained within a certain range of temperatures.

Typically, the cells (e.g. CHO cells) will initially be cultured at a“normal” temperature of 37.0° C.±1.0° C. until the target cell densityis achieved. The initial culture period is then followed by atemperature shift to the decreased temperature. After a period ofculturing at the decreased temperature, a temperature shift to thenormal temperature may or may not follow. Preferably, the cells (e.g.CHO cells) will initially be cultured at 37.0° C.±1.0° C. for severaldays, followed by manufacturing at a decreased temperature of 31.0-35.0°C.

Based on the present disclosure, those of ordinary skill in the art willbe able to select temperatures in which to grow cells, depending on theparticular needs of the respective cell line and the particularproduction requirements of the practitioner.

In certain embodiments, batch and fed-batch bioreactors are terminatedonce the expressed glycoprotein reaches a sufficiently high titer.Additionally or alternatively, batch and fed-batch bioreactors may beterminated once the cells reach a sufficiently high density, asdetermined by the needs of the practitioner. For example, the culturemay be terminated once the cells reach 1, 5, 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99 percent of maximalviable cell density. Additionally or alternatively, batch and fed-batchbioreactors may be terminated prior to excessive accumulation ofmetabolic waste products such as lactate and ammonium.

In certain cases, it may be beneficial to supplement a cell cultureduring the subsequent production phase with nutrients or other mediumcomponents that have been depleted or metabolized by the cells. Asnon-limiting examples, it may be beneficial to supplement a cell culturewith hormones and/or other growth factors, inorganic ions (such as, forexample, sodium, chloride, calcium, magnesium, and phosphate), buffers,vitamins, nucleosides or nucleotides, trace elements (inorganiccompounds usually present at very low final concentrations), aminoacids, lipids, or glucose or other energy source. Such supplementarycomponents may all be added to the cell culture at one time, or they maybe provided to the cell culture in a series of additions or they may beprovided together with fresh medium during a perfusion culture.

Alternatively to batch and fed-batch bioreactors the invention can alsobe practiced when cells expressing a glycoprotein of the invention arecultured in continuous perfusion bioreactors.

One of ordinary skill in the art will be able to tailor specific cellculture conditions in order to optimize certain characteristics of thecell culture including but not limited to growth rate, cell viability,final cell density of the cell culture, final concentration ofdetrimental metabolic byproducts such as lactate and ammonium, titer ofthe expressed glycoprotein, extent and composition of theoligosaccharide side chains or any combination of these or otherconditions deemed important by the practitioner.

Isolation of the Expressed Glycoprotein

In general, it will typically be desirable to isolate and/or purifyglycoproteins expressed according to the present invention. In certainembodiments, the expressed glycoprotein is secreted into the medium andthus cells and other solids may be removed, as by centrifugation orfiltering for example, as a first step in the purification process.

The expressed glycoprotein may be isolated and purified by standardmethods including, but not limited to, chromatography (e.g., ionexchange, affinity, size exclusion, and hydroxyapatite chromatography),gel filtration, centrifugation, or differential solubility, ethanolprecipitation and/or by any other available technique for thepurification of proteins (See, e.g., Scopes, Protein PurificationPrinciples and Practice 2nd Edition, Springer-Verlag, N.Y., 1987;Higgins, S. J. and Hames, B. D. (eds.), Protein Expression: A PracticalApproach, Oxford Univ Press, 1999; and Deutscher, M. P., Simon, M. I.,Abelson, J. N. (eds.), Guide to Protein Purification: Methods inEnzymology (Methods in Enzymology Series, Vol. 182), Academic Press,1997, each of which is incorporated herein by reference). Forimmunoaffinity chromatography in particular, the glycoprotein may beisolated by binding it to an affinity column comprising antibodies thatwere raised against that glycoprotein and were affixed to a stationarysupport. Alternatively, affinity tags such as an influenza coatsequence, poly-histidine, or glutathione-S-transferase can be attachedto the glycoprotein by standard recombinant techniques to allow for easypurification by passage over the appropriate affinity column. Proteaseinhibitors such as phenyl methyl sulfonyl fluoride (PMSF), leupeptin,pepstatin or aprotinin may be added at any or all stages in order toreduce or eliminate degradation of the glycoprotein during thepurification process. Protease inhibitors are particularly advantageouswhen cells must be lysed in order to isolate and purify the expressedglycoprotein. Additionally or alternatively, glycosidase inhibitors maybe added at any or all stages in order to reduce or eliminate enzymatictrimming of the covalently attached oligosaccharide chains. A preferredpurification method is described in Example 5 of this application.

Glycoproteins expressed according to the present invention have moreextensive sialylation than they would if grown under traditional cellculture conditions. Thus, one practical benefit of the present inventionthat may be exploited at the purification step is that the additionaland/or altered sialic acid residues on a glycoprotein grown inaccordance with certain of the present inventive methods may confer onit distinct biochemical properties that may be used by the practitionerto purify that glycoprotein more easily, or to a greater purity, thanwould be possible for a glycoprotein grown in accordance with moretraditional methods. For example, the glycoprotein can be purified orgreatly enriched by anion exchange chromatography, making use of thenegative charge of the sialic acid residues. Thereby a furtherenrichment of glycoprotein with high sialylation can be achieved.

In a further embodiment, the sialylation of the glycoprotein obtained bya method of the invention can be further increased by contacting theglycoprotein with a sialyltransferase in vitro. The sialyltransferasetypically is a mammalian sialyltransferase, preferably it is a humansialyltransferase. The sialyltransferase may be anα-2,3-sialyltransferase and/or an α-2,6-sialyltransferase. Preferably,the sialyltransferase is a human α-2,3-sialyltransferase (GenbankNP_775479-ST3GAL 1) and/or a human α-2,6-sialyltransferase. Mostpreferably, the sialyltransferase is human α-2,6-sialyltransferaseidentified by Genbank NP_003023-ST6GAL 1). Further present in the invitro reaction is a sialyl group donor, or sialic acid donor. Suitabledonors include, e.g., Cytidine-5′-monophospho-N-acetylneuraminic acid(CMP-NANA), Roche Catalog No. 05 974 003 103. A suitable kit for invitro sialylation is available from Roche (Catalog Number 07 012 250103).

One of ordinary skill in the art will appreciate that the exactpurification technique will vary depending on the character of theglycoprotein to be purified, the character of the cells from which theglycoprotein is expressed, and/or the composition of the medium in whichthe cells were grown.

As mentioned above, the invention, in a second aspect, relates to amethod of producing a glycoprotein comprising N-glycans with increasedsialylation, which comprises (i) providing cells comprising a nucleicacid encoding a polypeptide comprising a truncated von Willebrand Factor(VWF) and a recombinant nucleic acid encoding an α-2,3-sialyltransferaseand/or an α-2,6-sialyltransferase, preferably anα-2,6-sialyltransferase, and (ii) culturing the cells under conditionsthat allow expression of the glycoprotein.

The α-2,3-sialyltransferase preferably is a humanα-2,3-sialyltransferase. The cDNA sequence encoding humanα-2,3-sialyltransferase is shown in SEQ ID NO:12, and based thereon theskilled artisan can design suitable expression vectors containing acoding sequence of α-2,3-sialyltransferase.

The α-2,6-sialyltransferase preferably is a humanα-2,6-sialyltransferase. The cDNA sequence encoding humanα-2,6-sialyltransferase is shown in SEQ ID NO:7, and based thereon theskilled artisan can design suitable expression vectors containing acoding sequence of α-2,6-sialyltransferase.

The transfected cells can be cultured under conditions allowingexpression of the glycoprotein according to known culturing methods.

The glycoprotein can be recovered and/or isolated using establishedpurification techniques.

The embodiments described hereinabove in connection with the method ofthe first aspect of the invention apply to the method of the secondaspect mutatis mutandis.

Glycoprotein of the Invention

In a third aspect the invention relates to a glycoprotein obtainable bya method described herein.

In a fourth aspect, the invention relates to a glycoprotein comprising atruncated von Willebrand Factor (VWF), wherein said truncated VWF iscapable of binding to a Factor VIII

(FVIII), and wherein said glycoprotein comprises N-glycans, and at least75%, preferably at least 80%, more preferably at least 85% of saidN-glycans comprise, on average, at least one sialic acid moiety. Inpreferred embodiments, at least 87%, at least 90%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99%, of said N-glycans comprise, on average, atleast one sialic acid moiety. The inventors demonstrated thatpolypeptides comprising highly sialylated VWF fragments not only have aprolonged half-life themselves, but are also capable to extend thehalf-life of co-administered FVIII. In other words, administration ofthe glycoprotein of the invention leads to an extended half-life and/orto a reduced clearance of co-administered FVIII.

In a fifth aspect, the invention relates to a glycoprotein comprising atruncated von Willebrand Factor (VWF), wherein said truncated VWF iscapable of binding to a Factor VIII

(FVIII), and wherein said glycoprotein comprises N-glycans, wherein atleast 50% of the sialyl groups of the N-glycans of the glycoproteins areα-2,6-linked sialyl groups. In general, terminal sialyl groups can beattached to the galactose groups via a α-2,3- or via a α-2,6-linkage. Inone embodiment, N-glycans of the glycoprotein of the invention comprisemore α-2,6-linked sialyl groups than α-2,3-linked sialyl groups.Preferably, at least 60%, or at least 70%, or at least 80%, or at least90% of the sialyl groups of the N-glycans are α-2,6-linked sialylgroups. These embodiments can be obtained by, e.g., co-expressing humanα-2,6-sialyltransferase in mammalian cells.

In a sixth aspect, the invention relates to a glycoprotein comprising atruncated von Willebrand Factor (VWF), wherein said truncated VWF iscapable of binding to a Factor VIII (FVIII), and wherein saidglycoprotein comprises N-glycans, wherein at least 50% of the sialylgroups of the N-glycans of the glycoproteins are α-2,3-linked sialylgroups. In general, terminal sialyl groups can be attached to thegalactose groups via a α-2,3- or via a α-2,6-linkage. In one embodiment,N-glycans of the glycoprotein of the invention comprise moreα-2,3-linked sialyl groups than α-2,6-linked sialyl groups. Preferably,at least 60%, or at least 70%, or at least 80%, or at least 90% of thesialyl groups of the N-glycans are α-2,3-linked sialyl groups. Theseembodiments can be obtained by, e.g., co-expressing humanα-2,3-sialyltransferase in mammalian cells.

The preferred amino acid sequences of the glycoprotein of the inventionhave already been described hereinabove. The embodiments described abovein connection with the first aspect of the invention apply to the third,fourth, fifth and sixth aspects mutatis mutandis.

The “glycoprotein of the invention” as used herein refers to aglycoprotein according to the third, fourth, fifth or sixth aspect. Theglycoprotein of the invention has an increased sialylation of N-glycans,and in particular an increased α-2,6-sialylation or an increasedα-2,3-sialylation.

In one embodiment, at least 75%, at least 80%, at least 85%, at least90%, or at least 95% of the N-glycans of the glycoprotein of theinvention comprise at least one sialic acid group. In anotherembodiment, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99% ofthe N-glycans of the truncated VWF within the glycoprotein of theinvention comprise at least one sialic acid group.

In another embodiment, less than 25%, less than 20%, less than 15%, orless than 12%, or even less than 10%, or less than 8%, or less than 6%,or less than 5%, or less than 4%, or less than 3%, or less than 2% oreven less than 1% of the N-glycans of the glycoprotein of the inventionare asialo-N-glycans, i.e. they are N-glycans lacking a sialic acidgroup. In another embodiment, less than 25%, less than 20%, less than15%, or less than 12%, or less than 10%, or less than 8%, or less than6%, or less than 5%, or less than 4%, or less than 3%, or less than 2%or even less than 1% of the N-glycans of the truncated VWF within theglycoprotein of the invention are asialo-N-glycans, i.e. they do nothave a sialic acid group.

In another embodiment, at least 30%, or at least 35%, or at least 40% ofthe N-glycans of the glycoprotein of the invention aremonosialo-N-glycans, i.e. they are N-glycans with one sialic acid group.In another embodiment, at least 30%, or at least 35%, or at least 40% ofthe N-glycans of the truncated VWF within the glycoprotein of theinvention are monosialo-N-glycans. By way of non-limiting example theamount of monosialylated N-glycans can be determined as detailed inExample 6 and in Example 12.

In yet another embodiment, at least 15%, or at least 25%, or at least30% of the N-glycans of the glycoprotein of the invention aredisialo-N-glycans, i.e. they are N-glycans with 2 sialic acid groups. Inyet another embodiment, at least 15%, or at least 25%, or at least 30%of the N-glycans of the truncated VWF within the glycoprotein of theinvention are disialo-N-glycans. By way of non-limiting example theamount of disialylated N-glycans can be determined as detailed inExample 6 and in Example 12.

In yet another embodiment, at least 5%, or at least 10%, of theN-glycans of the glycoprotein of the invention are trisialo-N-glycans,i.e. they are N-glycans with 3 sialic acid groups. In yet anotherembodiment, at least 5%, or at least 10%, of the N-glycans of thetruncated VWF within the glycoprotein of the invention aretrisialo-N-glycans. By way of non-limiting example the amount oftrisialylated N-glycans can be determined as detailed in Example 6 andin Example 12.

In another embodiment, at least 20%, or at least 30%, or at least 40%,of the N-glycans of the glycoprotein of the invention comprise two ormore sialic acid groups. In another embodiment, at least 20%, or atleast 30%, or at least 40%, of the N-glycans of the truncated

VWF within the glycoprotein of the invention comprise two or more sialicacid groups.

Other preferred embodiments of the invention comprise a truncated vonWillebrand Factor (VWF), wherein said truncated VWF is capable ofbinding to a Factor VIII (FVIII), and wherein said glycoproteincomprises N-glycans, wherein less than 35%, preferably less than 34%,preferably less than 33%, preferably less than 32%, preferably less than31%, preferably less than 30%, preferably less than 29%, preferably lessthan 28%, preferably less than 27% preferably less than 26%, preferablyless than 25%, preferably less than 24%, preferably less than 23%,preferably less than 22%, preferably less than 21%, preferably less than20%, preferably less than 19%, preferably less than 18%, preferably lessthan 17%, preferably less than 16%, preferably less than 15%, preferablyless than 14%, preferably less than 13%, preferably less than 12%,preferably less than 11%, preferably less than 10%, preferably less than9%, preferably less than 8%, preferably less than 7%, preferably lessthan 6% and preferably less than 5% of said N-glycans comprise, onaverage, two or more terminal and non-sialylated galactose residues.

Still other even more preferred embodiments of the invention comprise atruncated von Willebrand Factor (VWF), wherein said truncated VWF iscapable of binding to a Factor VIII (FVIII), and wherein said truncatedVWF comprises N-glycans, wherein less than 6%, preferably less than 5%,preferably less than 4%, preferably less than 3%, preferably less than2%, and preferably less than 1% of said N-glycans comprise, on average,three or more terminal and non-sialylated galactose residues.

The above-described embodiments can be combined with each other. Anypercentages of N-glycans mentioned above, or any indications of thedegree of sialylation, are to be understood as average percentages ordegrees, i.e. they refer to a population of molecules, not to a singlemolecule. It is clear that the glycosylation or sialylation of theindividual glycoprotein molecules within a population of glycoproteinswill show some heterogeneity.

It has further been found that the glycoproteins obtained in accordancewith this invention have a high proportion of dimers. The glycoproteinof the invention is therefore preferably present as dimer. In oneembodiment, at least 50%, or at least 60%, or at least 70%, or at least80%, or at least 90%, or at least 95% or about 100% of the glycoproteinsare present as dimers. In another embodiment, the ratio dimer:monomer ofthe glycoprotein of the invention is at least 1.5, preferably at least2, more preferably at least 2.5 or at least 3. The dimer formationobtained by the methods of the invention is favorable, as the dimer hasan improved affinity to Factor VIII. The dimer content, and the ratio ofdimer to monomer of the glycoprotein of the invention can be determinedas described in Example 5.

In another preferred embodiment, the glycoprotein of the inventioncomprises a heterologous polypeptide, e.g. a HLEP as defined above. Mostpreferably, the HLEP is human serum albumin (see SEQ ID NO:11). Theembodiments described supra apply here mutatis mutandis.

The glycoprotein of the invention is preferably capable of binding toFactor VIII (see above). In one embodiment, the affinity of theglycoprotein of the invention to Factor VIII is greater than that ofhuman native VWF to the same Factor VIII. The factor VIII affinity mayrefer to human native Factor VIII, or to the Factor VIII characterizedby SEQ ID NO:10.

It has been found that preparations of the glycoprotein in accordancewith this invention with a high proportion of dimers do have anincreased affinity to Factor VIII. Such increased affinity to FactorVIII does lead to an enhanced stabilization of Factor VIII by theglycoproteins of the present invention. Alternatively to or incombination with an increased dimer proportion also glycoproteins inaccordance with the invention with mutations within the Factor VIIIbinding domain which do increase the affinity to Factor VIII arepreferred embodiments of the invention. Suitable mutations aredisclosed, e.g., in WO 2013/120939 A1.

Another aspect of the invention is a glycoprotein as defined herein foruse in the treatment of a blood coagulation disorder. The treatmentcomprises administering to a patient an effective amount of theglycoprotein. The treatment may further comprise administering a FVIII.

Another aspect of the invention is a pharmaceutical compositioncomprising a glycoprotein of the invention, and a pharmaceuticallyacceptable excipient or carrier.

Another aspect of the present invention is a pharmaceutical kitcomprising (i) a glycoprotein as defined hereinabove and (ii) a FactorVIII. Preferably, the glycoprotein and the FVIII are contained inseparate compositions.

Another aspect of the present invention is a pharmaceutical kitcomprising (i) a glycoprotein as defined hereinabove and (ii) a FactorVIII, for simultaneous, separate or sequential use in the treatment of ablood coagulation disorder.

Another aspect of the invention is the use of a polypeptide as definedhereinabove for increasing the terminal half-life or mean residence time(MRT) or reducing the clearance of Factor VIII. For evaluation of thepharmacokinetic data a linear pharmacokinetics model (compoundelimination via the central compartment) was applied. Accordingly, anypharmacokinetic parameters used herein are based on a linearpharmacokinetics model (compound elimination via the centralcompartment), unless indicated otherwise.

The “half-life” T½(t) at a certain time t is the time it takes to halvethe plasma concentration C(t) that is present at time t, i.e.C[t+T½(t)]=C(t)/2. The “terminal half-life” is the limit of T½(t) when ttends to infinity. The area under the curve (AUC) can be determined toassess clearance effects. A reduction in clearance leads to higher AUCvalues, and to an increase in half-life.

The term “MRT”, as used herein, means the average time a drug moleculeresides in the body. In a linear pharmacokinetic system with constantclearance MRT can be calculated as area under the first moment curve(AUMC) divided by AUC. The first moment curve is time multiplied byplasma concentration at that time.

The MRT of administered FVIII is increased by at least 25%, preferablyby at least 50%, more preferably by at least 75%, more preferably by atleast 100%, most preferably by at least 125%, if an effective amount ofthe glycoprotein of the present invention is co-administered, i)relative to administration of the FVIII alone or ii) relative toadministration of a reference protein which has the same proteinsequence as the glycoprotein of the invention but a completelydesialylated N-glycan structure or iii) relative to administration of areference protein which has the same protein sequence as theglycoprotein of the invention but more than 35% of its N-glycanscomprise two or more terminal and non-sialylated N-glycans and more than6% of its N-glycans comprise three or more terminal and non-sialylatedgalactose residue.

The MRT of the glycoprotein prepared according to the method of thepresent invention comprising culturing at a reduced temperature isgreater than that of a reference glycoprotein having the same amino acidsequence which was cultured at 37° C. The increase in MRT of theglycoprotein prepared according to the method of the present invention(or of any glycoprotein of the present invention) relative to thereference glycoprotein is preferably at least 25%, more preferably atleast 50%, most preferably at least 100%.

The term “clearance”, as used herein, refers to the rate at which plasmais cleared of drug. Specifically, it is the current elimination rate ofa drug divided by its current plasma concentration. In a linearpharmacokinetic system after a single intravenous administration theclearance can be calculated as the ratio of dose over the area under theplasma concentration-time curve (AUC), provided the clearance isconstant. The lower the clearance the longer it takes until the plasmais cleared of the drug.

The clearance of administered FVIII is reduced by at least 10%,preferably by at least 25%, more preferably by at least 40%, morepreferably by at least 50%, most preferably by at least 60%, if aneffective amount of the glycoprotein of the present invention isco-administered, i) relative to administration of the FVIII alone or ii)relative to administration of a reference protein which has the sameprotein sequence as the glycoprotein of the invention but a completelydesialylated N-glycan structure or iii) relative to administration of areference protein which has the same protein sequence as theglycoprotein of the invention but more than 35% of its N-glycanscomprise two or more terminal and non-sialylated N-glycans and more than6% of its N-glycans comprise three or more terminal and non-sialylatedgalactose residue.

The clearance of the glycoprotein prepared according to the method ofthe present invention comprising culturing at a reduced temperature islower than that of a reference glycoprotein having the same amino acidsequence which was cultured at 37° C. The reduction in clearance of theglycoprotein prepared according to the method of the present invention(or of any glycoprotein of the present invention) relative to thereference glycoprotein is preferably at least 40%, more preferably atleast 50%, most preferably at least 60%.

The invention further relates to a method of increasing the MRT orhalf-life, or to a method of reducing the clearance of Factor VIII invivo, comprising administering to a subject an effective amount of aglycoprotein as defined hereinabove.

A further aspect of this invention is a method of treating a bloodcoagulation disorder, comprising administering to a patient in needthereof an effective amount of a glycoprotein as defined hereinabove.

A further aspect is the use of a glycoprotein as defined hereinabove forreducing the frequency of administration of FVIII in a treatment ofhemophilia A. The frequency of intravenous or subcutaneousadministration of FVIII may be reduced to twice per week. Alternatively,the frequency of intravenous or subcutaneous administration of FVIII maybe reduced to once per week, or even lower, e.g. to once per 10 days oronce per 14 days. The FVIII may be administered twice weekly, every 5days, once weekly, every 10 days, every two weeks, every three weeks,every four weeks or once a month, or in any range between any two of theforegoing values, for example from every four days to every month, fromevery 10 days to every two weeks, or from two to three times a week,etc.

Another aspect is the use of a glycoprotein as defined hereinabove forreducing the dose of FVIII to be administered in a treatment ofhemophilia A.

Treatment of Coagulation Disorder

The glycoproteins of the invention are useful for treating coagulationdisorders including hemophilia A. The term “hemophilia A” refers to adeficiency in functional coagulation FVIII, which is usually inherited.

Treatment of a disease encompasses the treatment of patients alreadydiagnosed as having any form of the disease at any clinical stage ormanifestation; the delay of the onset or evolution or aggravation ordeterioration of the symptoms or signs of the disease; and/or preventingand/or reducing the severity of the disease.

A “subject” or “patient” to whom a glycoprotein of the invention isadministered preferably is a human. In certain aspects, the human is apediatric patient. In other aspects, the human is an adult patient.

Compositions comprising a glycoprotein of the invention and, optionallyone or more additional therapeutic agents, such as the secondtherapeutic agents described below, are described herein. Thecompositions typically are supplied as part of a sterile, pharmaceuticalcomposition that includes a pharmaceutically acceptable carrier. Thiscomposition can be in any suitable form (depending upon the desiredmethod of administering it to a patient).

The glycoproteins of the invention can be administered to a patient by avariety of routes such as orally, transdermally, subcutaneously,intranasally, intravenously, intraperitoneally, intramuscularly,intrathecally, topically or locally. The most suitable route foradministration in any given case will depend on the particularglycoprotein, the subject, and the nature and severity of the diseaseand the physical condition of the subject. Typically, a glycoprotein ofthe invention will be administered intravenously.

The glycoprotein and the FVIII are preferably administered intravenouslyor subcutaneously.

In a first embodiment, both the glycoprotein and the FVIII areadministered intravenously. In a second embodiment, both theglycoprotein and the FVIII are administered subcutaneously.

In another embodiment, the FVIII is administered intravenously, and theglycoprotein is administered via a different route. In furtherembodiments, the glycoprotein is administered subcutaneously, and theFVIII is administered via a different route. For example, theglycoprotein may be administered subcutaneously, and the FVIII may beadministered intravenously.

In further embodiments, the FVIII is administered subcutaneously, andthe glycoprotein is administered via a different route. In furtherembodiments, the glycoprotein is administered intravenously, and theFVIII is administered via a different route. For example, theglycoprotein may be administered intravenously, and the FVIII may beadministered subcutaneously.

The term “Factor VIII” and “FVIII” are used interchangeably herein andencompass both plasma derived FVIII and recombinant FVIII. RecombinantFVIII encompasses without limitation full-length FVIII as well astwo-chain B-domain deleted or truncated variants as well as single-chainB-domain deleted or truncated variants for example those described in WO2004/067566 and other FVIII variants with mutations outside the B-domainbut having the biological activity of FVIII.

Determination of the effective dosage, total number of doses, and lengthof treatment with a glycoprotein of the invention is well within thecapabilities of those skilled in the art, and can be determined using astandard dose escalation study.

Pharmaceutical Compositions

Therapeutic formulations of the glycoproteins of the invention suitablein the methods described herein can be prepared for storage aslyophilized formulations or aqueous solutions by mixing the glycoproteinhaving the desired degree of purity with optionalpharmaceutically-acceptable carriers, excipients or stabilizerstypically employed in the art (all of which are referred to herein as“carriers”), i.e., buffering agents, stabilizing agents, preservatives,isotonifiers, non-ionic detergents, antioxidants, and othermiscellaneous additives. See, Remington's Pharmaceutical Sciences, 16thedition (Osol, ed. 1980). Such additives must be nontoxic to therecipients at the dosages and concentrations employed.

Buffering agents help to maintain the pH in the range which approximatesphysiological conditions. They can present at concentration ranging fromabout 2 mM to about 50 mM. Suitable buffering agents include bothorganic and inorganic acids and salts thereof such as citrate buffers(e.g., monosodium citrate-disodium citrate mixture, citricacid-trisodium citrate mixture, citric acid-monosodium citrate mixture,etc.), succinate buffers (e.g., succinic acid- monosodium succinatemixture, succinic acid-sodium hydroxide mixture, succinic acid- disodiumsuccinate mixture, etc.), tartrate buffers (e.g., tartaric acid-sodiumtartrate mixture, tartaric acid-potassium tartrate mixture, tartaricacid-sodium hydroxide mixture, etc.), fumarate buffers (e.g., fumaricacid-monosodium fumarate mixture, fumaric acid-disodium fumaratemixture, monosodium fumarate-disodium fumarate mixture, etc.), gluconatebuffers (e.g., gluconic acid-sodium glyconate mixture, gluconicacid-sodium hydroxide mixture, gluconic acid-potassium glyuconatemixture, etc.), oxalate buffer (e.g., oxalic acid-sodium oxalatemixture, oxalic acid-sodium hydroxide mixture, oxalic acid-potassiumoxalate mixture, etc), lactate buffers (e.g., lactic acid-sodium lactatemixture, lactic acid-sodium hydroxide mixture, lactic acid-potassiumlactate mixture, etc.) and acetate buffers (e.g., acetic acid-sodiumacetate mixture, acetic acid-sodium hydroxide mixture, etc.).Additionally, phosphate buffers, histidine buffers and trimethylaminesalts such as Tris can be used.

Preservatives can be added to retard microbial growth, and can be addedin amounts ranging from 0.2%-1% (w/v). Suitable preservatives includephenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben,octadecyldimethylbenzyl ammonium chloride, benzalconium halides (e.g.,chloride, bromide, and iodide), hexamethonium chloride, and alkylparabens such as methyl or propyl paraben, catechol, resorcinol,cyclohexanol, and 3-pentanol. Isotonicifiers sometimes known as“stabilizers” can be added to ensure isotonicity of liquid compositionsand include polhydric sugar alcohols, preferably trihydric or highersugar alcohols, such as glycerin, erythritol, arabitol, xylitol,sorbitol and mannitol. Stabilizers refer to a broad category ofexcipients which can range in function from a bulking agent to anadditive which solubilizes the therapeutic agent or helps to preventdenaturation or adherence to the container wall. Typical stabilizers canbe polyhydric sugar alcohols (enumerated above); amino acids such asarginine, lysine, glycine, glutamine, asparagine, histidine, alanine,ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc.,organic sugars or sugar alcohols, such as lactose, trehalose, stachyose,mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glyceroland the like, including cyclitols such as inositol; polyethylene glycol;amino acid polymers; sulfur containing reducing agents, such as urea,glutathione, thioctic acid, sodium thioglycolate, thioglycerol,α-monothioglycerol and sodium thio sulfate; low molecular weightpolypeptides (e.g., peptides of 10 residues or fewer); proteins such ashuman serum albumin, bovine serum albumin, gelatin or immunoglobulins;hydrophylic polymers, such as polyvinylpyrrolidone monosaccharides, suchas xylose, mannose, fructose, glucose; disaccharides such as lactose,maltose, sucrose and trisaccacharides such as raffinose; andpolysaccharides such as dextran. Stabilizers can be present in the rangefrom 0.1 to 10,000 weights per part of weight active protein.

Non-ionic surfactants or detergents (also known as “wetting agents”) canbe added to help solubilize the therapeutic agent as well as to protectthe therapeutic protein against agitation-induced aggregation, whichalso permits the formulation to be exposed to shear surface stressedwithout causing denaturation of the protein. Suitable non-ionicsurfactants include polysorbates (20, 80, etc.), polyoxamers (184, 188etc.), Pluronic polyols, polyoxyethylene sorbitan monoethers (TWEEN®-20,TWEEN®-80, etc.). Non-ionic surfactants can be present in a range ofabout 0.05 mg/ml to about 1.0 mg/ml, or in a range of about 0.07 mg/mlto about 0.2 mg/ml.

Additional miscellaneous excipients include bulking agents (e.g.,starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbicacid, methionine, vitamin E), and cosolvents. The formulation herein canalso contain a second therapeutic agent in addition to a glycoprotein ofthe invention. Examples of suitable second therapeutic agents areprovided below.

The dosing schedule can vary from once a month to daily depending on anumber of clinical factors, including the type of disease, severity ofdisease, and the patients sensitivity to the glycoprotein of theinvention. In specific embodiments, a glycoprotein of the invention isadministered, twice weekly, every 5 days, once weekly, every 10 days,every two weeks, every three weeks, every four weeks or once a month, orin any range between any two of the foregoing values, for example fromevery four weeks to every month, from every 10 days to every two weeks,or from two to three times a week, etc.

The dosage of a glycoprotein of the invention to be administered willvary according to the particular glycoprotein, the subject, and thenature and severity of the disease, the physical condition of thesubject, the therapeutic regimen (e.g., whether a second therapeuticagent is used), and the selected route of administration; theappropriate dosage can be readily determined by a person skilled in theart.

It will be recognized by one of skill in the art that the optimalquantity and spacing of individual dosages of a glycoprotein of theinvention will be determined by the nature and extent of the conditionbeing treated, the form, route and site of administration, and the ageand condition of the particular subject being treated, and that aphysician will ultimately determine appropriate dosages to be used. Thisdosage can be repeated as often as appropriate. If side effects developthe amount and/or frequency of the dosage can be altered or reduced, inaccordance with normal clinical practice.

Combination Therapy

Preferably, the patient being treated with the glycoprotein of theinvention is also treated with a conventional therapy of coagulationdisorders. For example, a patient suffering from hemophilia is typicallyalso being treated with Factor VIII.

In accordance with this invention, the patient being treated with theglycoprotein of the invention is also treated with Factor VIII. Theglycoprotein of the invention and the Factor VIII molecule may beadministered simultaneously or in a sequential fashion, both modes ofadministration being encompassed by the term “combination therapy” and“co-administration”. The glycoprotein of the invention and the FactorVIII molecule may be administered as a mixture, i.e. within the samecomposition, or separately, i.e. as separate compositions.

The concentration of Factor VIII in the composition used according tothe present invention is typically in the range of 10-10,000 IU/mL. Indifferent embodiments, the concentration of FVIII in the compositions ofthe invention is in the range of 10-8,000 IU/mL, or 10-5,000 IU/mL, or20-3,000 IU/mL, or 50-1,500 IU/mL, or 3,000 IU/mL, or 2,500 IU/mL, or2,000 IU/mL, or 1,500 IU/mL, or 1,200 IU/mL, or 1,000 IU/mL, or 800IU/mL, or 750 IU/mL, or 600 IU/mL, or 500 IU/mL, or 400 IU/mL, or 300IU/mL, or 250 IU/mL, or 200 IU/mL, or 150 IU/mL, or 125 IU/mL, or 100IU/mL, or 62.5 IU/mL, or 50 IU/mL.

“International Unit,” or “IU,” is a unit of measurement of the bloodcoagulation activity (potency) of FVIII as measured by a FVIII activityassay such as a one stage clotting assay or a chromogenic substrateFVIII activity assay using a standard calibrated against aninternational standard preparation calibrated in “IU”. One stageclotting assays are known to the art, such as that described in N Lee,Martin L, et al., An Effect of Predilution on Potency Assays of FVIIIConcentrates, Thrombosis Research (Pergamon Press Ltd.) 30, 511 519(1983). Principle of the one stage assay: The test is executed as amodified version of the activated Partial Thromboplastin Time(aPTT)-assay: Incubation of plasma with phospholipids and a surfaceactivator leads to the activation of factors of the intrinsiccoagulation system. Addition of calcium ions triggers the coagulationcascade. The time to formation of a measurable fibrin clot isdetermined. The assay is executed in the presence of Factor VIIIdeficient plasma. The coagulation capability of the deficient plasma isrestored by Coagulation Factor VIII included in the sample to be tested.The shortening of coagulation time is proportional to the amount ofFactor VIII present in the sample. The activity of Coagulation FactorVIII is quantified by direct comparison to a standard preparation with aknown activity of Factor VIII in International Units.

Another standard assay is a chromogenic substrate assay. Chromogenicsubstrate assays may be purchased commercially, such as the coamaticFVIII test kit (Chromogenix-Instrumentation Laboratory SpA V. le Monza338-20128 Milano, Italy). Principle of the chromogenic assay: In thepresence of calcium and phospholipid, Factor X is activated by FactorIXa to Factor Xa. This reaction is stimulated by Factor Villa ascofactor. FVIIIa is formed by low amounts of thrombin in the reactionmixture from FVIII in the sample to be measured. When using the optimumconcentrations of Ca2+, phospholipid and Factor IXa and an excessquantity of Factor X, activation of Factor X is proportional to thepotency of Factor VIII. Activated Factor X releases the chromophore pNAfrom the chromogenic substrate S-2765. The release of pNA, measured at405 nm, is therefore proportional to the amount of FXa formed, and,therefore, also to the Factor VIII activity of the sample.

The nucleotide and amino acid sequences shown in the sequence listingare summarized in the following table:

TABLE 1 SEQ ID NO: Remarks 1 DNA sequence encoding a polypeptidecomprising amino acids 1 to 1242 of human VWF, a glycine/serine linkerand human albumin; nucleotide positions (nt): nt 1-6: EcoRI restrictionenzyme cleavage site nt 32-3757: coding sequence for VWF amino acids 1to 1242 nt 3758-3850: coding sequence for glycine/serine linker nt3851-5608: coding sequence for human albumin nt 5609-5616: Notlrestriction enzyme cleavage site 2 Amino acid sequence encoded by SEQ IDNO: 1 (mature form): amino acid positions (aa): aa 1-479: VWF D'D3region (VWF amino acids 764-1242) aa 480-510: glycine/serine linker aa511-1195: human albumin 3 PCR primer α-2,6 sialyltransferase 4 PCRprimer α-2,6 sialyltransferase 5 nested PCR primer α-2,6sialyltransferase 6 nested PCR primer α-2,6 sialyltransferase 7 cDNAsequence encoding human α-2,6 sialyltransferase 8 DNA sequence encodingthe pre-pro form of human native VWF 9 Amino acid sequence encoded bySEQ ID NO: 8 10 Amino acid sequence of a single chain Factor VIIImolecule 11 Amino acid sequence of mature human serum albumin 12 cDNAsequence encoding human α-2,3 sialyltransferase

The following examples illustrate the invention but should not beconstrued as limiting the present invention to the specific embodimentsdescribed herein below.

Example 1: Generation of D′D3 Albumin Fusion Protein (D′D3-FP)

The expression cassette for D′D3-FP consisting of cDNA encoding VWFamino acids 1 to 1242, a glycine/serine linker and the cDNA of humanalbumin was prepared by custom gene synthesis (Eurofins Genomics,Ebersberg, Germany). Through flanking restriction sites (EcoRI, NotI)the expression cassette was excised from the cloning vector supplied andinserted into a pIRESneo3 vector (BD Biosciences, Franklin Lakes, N.J.,USA) linearized with EcoRI and NotI. The resulting expression plasmidcontained nucleotide sequences encoding the VWF propeptide, D′ and D3(VWF amino acids 1 to 1242 of SEQ ID NO:9) fused to the albumin codingsequence through a short linker coding sequence under CMV promotercontrol. The nucleotide sequence of the coding sequence is displayed asSEQ ID NO:1, the amino acid sequence of the mature D′D3-FP is shown asSEQ ID NO:2.

Example 2: Transfection of Plasmids and Stable Expression of D′D3-FPDimer in Chinese Hamster Ovary (CHO) Cells

The expression plasmid as described above was grown up in XL10 Gold(Agilent Technologies) and purified using standard protocols (Qiagen,Hilden, Germany).

CHO K1 cells were transfected using the Lipofectamine 2000 reagent(Invitrogen) and grown up in serum-free medium (CD-CHO, Invitrogen) inthe presence of 500-1000 μg/ml Geneticin. An expression plasmid encodingPACE/furin (pFu-797) as described in WO2007/144173 was cotransfected tomaximize propeptide cleavage efficacy. Single cell derived clones weregrown up and selected according to their D′D3-FP expression yield asquantified by an albumin specific enzyme immunoassay (see below). Thecell line finally selected for D′D3-FP fermentation was calledT2050-CL3.

Example 3: Coexpression of an α-2,6 Sialyl Transferase

During a cell line generation process as described in example 2 aplasmid carrying an expression unit encoding an α-2,6 sialyl transferaseto support the attachment of non-rodent sialic acids can becotransfected.

The coding sequence for human α-2,6 sialyl transferase is amplified froma human liver cDNA library (Ambion) using primers We2556 (SEQ-ID NO. 3)and We 2558 (SEQ-ID NO. 4) for a first and primers We2553 (SEQ-ID NO. 5)and We 2559 (SEQ-ID NO. 6) for a second PCR in a nested PCR setup. Forthe first PCR 2 μL of the Ambion human liver cDNA library are mixed with34.5 μL of water, 10 μl 5× PCR buffer Phusion GC (New England Biolabs),1 μl of 10 mM dNTPs, 1 μL of We2556 (10 pmol), 1 μl of We2558 (10 pmol)and 0.5 μL of Phusion DNA polymerase (New England Biolabs) and amplifiedusing a touchdown protocol of initial 60 seconds at 98° C., 15 cycles ofa) 15 seconds of denaturation at 98° C., b) 30 seconds of annealing at64° C. and c) 2 minutes of elongation at 72° C., wherein the temperatureof the annealing step is reduced by 0.3° C. per cycle, followed by 25cycles of a) 25 seconds of denaturation at 98° C., b) 30 seconds ofannealing at 62° C. and c) 2 minutes of elongation at 72° C., followedby a final extension step for 10 minutes at 72° C., after which thereaction is stopped by cooling down and holding at 4° C. For the nestedPCR 2 μL of the first PCR reaction are mixed with 34.5 μL of water, 10μl 5× PCR buffer Phusion GC, 1 μl of 10 mM dNTPs, 1 μL of We2553 (10pmol), 1 μl of We2559 (10 pmol) and 0.5 μL of Phusion DNA polymerase andamplified using the touchdown protocol as described for the first PCR.The nested PCR adds an NheI restriction enzyme cutting site to the5′-end and a BamH1 site to the 3′-end of the PCR fragment. This fragmentis cut by NheI and BamH1 and ligated into expression vector plRESneo3which had been opened by the same enzymes. The resulting expressionvector then can be used for cotransfection.

Example 4: Production of D′D3-FP in Bioreactors

The fermentation process for the production of D′D3-FP started with thethaw of cell line T2050-CL3 followed by cell expansion in shake flasksand finally a fermentation process in perfusion mode using the SartoriusBioStat B-DCU 5 L bioreactor and the BioStat STR 50L single-usebioreactors. The BioSeps 10L or 200L (Applikon), respectively, were usedas cell retention devices. Cell culture media were either PowerCHO3(Lonza BESP1204) with 8 mM L-glutamine and 1 μM CuSO₄ or ProCHO5 (LonzaBESP1072) with 10 mM L-glutamine and 1 μM CuSO₄.

The seed trains in shake flasks were performed at 37° C., 7.5% CO₂ at ashaker speed of 160 rpm.

The 5L bioreactor was inoculated with a target VCD of 2.5×10⁵ cells/mL.The cells were cultivated in PowerCHO3 with 8 mM L-glutamine and 1 μMCuSO₄ at a temperature of +37.0° C., a pH of 7.00, and at 30% oxygensaturation. A temperature shift to +34.0 ° C. (evaluated range +31° C.to +35° C.) was performed after initial harvests from the bioreactor runat +37° C. had been taken. The pH was controlled using CO₂ sparged asacid and

NaHCO₃ as base. The overlay air flow rate was set to 0.5 L/min. A ringsparger was used as a sparging unit. The agitation rate was 150 rpm witha 2fold pitch blade impeller in down pull mode.

The 50 L bioreactor was inoculated with a target VCD of 3.0×10⁵cells/mL. The cells were cultivated in ProCHO5 medium with 10 mML-glutamine and 1 μM CuSO₄ at a temperature of +37.0° C., a pH of 6.90,and at 30% oxygen saturation. A temperature shift to +34.0 ° C. wasperformed after the initial one or two harvests. PH control as above,the overlay air flow rate was set to 2 L/min. A micro sparger was usedas a sparging unit. The agitation rate was 90 rpm with a 2fold pitchblade impeller in down pull mode.

The perfusion was initiated when the VCD in the bioreactor was ≥1.0×10⁶cells/mL. The perfusion rate was set to 1.0 volume/volume/day. TheBioSep was operated in back flush mode with 5 (10) minutes runtime and10 seconds back flush at a power input of 7 (30) W (numbers in bracketsrefer to the 50 L bioreactor). The perfusate and the bleed were filteredinline and collected in bags over 48 hours at +2 to +8° C. The VCD wascontrolled by active bleeding using a turbidity probe using glucoseconsumption as parameter with a target of 2 g/L glucose. Harvest andbleed were filtered inline, the harvest system consisting of adisposable filter and disposable bag was changed every second day.

To prepare material for the PK analyses described below harvests takenbefore and after the respective temperature shifts were purified byaffinity and size exclusion chromatography.

Example 5: Purification of D′D3-FP Dimer Using Affinity Chromatographyand Size Exclusion Chromatography

The cell-free harvest from the bioreactor was concentrated 30-fold usinga TFF system (e.g. Pall Centramate 500 S) with a 30 kD membrane (e.gPall Centramate OS030T12). That concentrate was spiked with NaCl andEDTA to a final concentration of 0.75 M NaCl and 5 mM EDTA and loadedovernight on a CaptureSelect Human Albumin column (Life Technologies)which was pre-equilibrated with 20 mM Tris buffer pH 7.4. After washingthe column with equilibration buffer D′D3-FP was eluted with elutionbuffer (20 mM Tris, 2 M MgCl₂, pH 7.4). The eluate was then 10-foldconcentrated and dialyzed against 50 mM Tris, 150 mM NaCl, pH 7.4 usingUltra Centrifugal Filters with a 30 kD cut-off (e.g. Amicon. UFC903024).To separate the D′D3-FP dimer from the monomer portion that material wasloaded on a Superdex 200 pg column (GE Healthcare Code: 17-1069-01)pre-equilibrated with 50 mM Tris, 150 mM NaCl, pH 7.4 and the peakfractions containing the D′D3-FP dimer were pooled. The area under thecurve for the dimer and monomer peak fractions were used to calculatedimer to monomer ratio.

Example 6: Total Sialylation Assay

Materials and Methods:

Acetic acid was from Sigma-Aldrich (Prod. 338826). Acetonitrile was fromBurdick and Jackson (Prod. LC015). 2-aminobenzamide (2-AB) was fromAldrich (Prod. A89804). Ammonium hydroxide was from Sigma-Aldrich (Prod.338818). Ammonium bicarbonate was from Fluka (Prod. 09830). Dimethylsulfoxide was from Sigma Prod. (D2650). Dithiothreitol (DTT) was fromSigma (Prod.646563). Formic acid was from Thermo (Prod. 28905).N-Glycosidase F (PNGase 250U) was from Roche (Prod. 11 365 193 00).Sodium cyanoborohydride was from Aldrich (Prod. 156159). Oasis HLB 3cc60 mg SPE cartridges were from Waters (Part No: WAT094226). 50 KDaAmicon Ultra 4 centrifugal ultrafilters were from Millipore (Cat. No.UFC805008). Zeba Spin 7K MWCO columns 2 mL were from Thermo (No. 89889)

PNGase F Enzymatic Glycan Release:

About 700 μg of D′D3-FP was reduced with DTT in approximately 70 mMammonium bicarbonate, pH 8.5 at 60° C. for 30 min. The reduced samplewas cooled to room temperature and alkylated with iodoacetamide at RT inthe dark for 30 min. The alkylated sample was buffer exchanged into 50mM ammonium bicarbonate pH 8.6 using a 2 mL Zeba Spin 7K MWCO column. Tothe buffer exchanged sample, 40U of PNGase was added and the sampleincubated at 37° C. for 14 hours. An additional 40U of PNGase was addedand the sample incubated for a further 6 hours at 37° C. The PNGasedigested sample was centrifuged through a 50 KDa Amicon Ultra 4ultrafilter. The filtrate was dried in a CentriVap.

2-AB Labelling of Released N-Glycans:

To prepare the 2-AB labelling reagent, 23 mg of 2-aminobenzamide wasdissolved in 350 μL of DMSO and 150 μL glacial acetic acid was added.The resulting solution was added to 32 mg of sodium cyanoborohydride andmixed thoroughly until dissolved. 50 μL of the 2-AB reagent was added tothe dried sample and incubated in the dark at 65° C. for 3.5 hours.

A Waters Oasis HLB 3cc 60 mg SPE cartridge was conditioned with 3 mL 95%acetonitrile the 3 mL 35% acetonitrile then 3 mL 95% acetonitrile. The2-AB labelled sample was diluted by adding 1.95 mL of 95% v/vacetonitrile and immediately loaded onto the HLB cartridge and allowedto drain under gravity. Sample was washed under gravity with 3×3 mL of95% v/v acetonitrile and the eluted with 3 mL of 35% v/v acetonitrile.The eluate was dried in a Centrivap. The dried 2-AB derivatised samplewas dissolved by the addition of 35 μL of Milli Q water and vortexmixing. After dissolution, 85 μL of Acetonitrile was added and mixedbriefly. Sample was transferred to a HPLC vial for analysis.

2-AB Glycan Analysis:

High performance liquid chromatography was performed on a Thermo DionexUltimate 3000 system consisting of an RS Binary Pump, Autosampler, RSColumn Compartment and RS Fluorescence detector. The separation of 2-ABglycan derivatives was achieved using a Dionex GlycanPac AXH-1, 1.9 μm,2.1×150 mm column (P/N 082472). Mobile phase A consisted of 100%acetonitrile, Mobile phase B consisted of 50 mM Formic acid adjusted topH 4.0 with 5M ammonium hydroxide solution. The column was maintained at50° C. and the flow rate was 0.200 mL/min. The column was equilibratedwith 15% B. After injection of 6 μL of sample, the mobile phasecomposition was changed linearly to 40% B over 50 minutes, then to 80% Bover 10 minutes, then to 95% B over 0.1 minutes, then maintained at 95%B for 4.9 minutes, and then back to 15% B over 0.1 minutes. The columnwas requilibrated at 15% B for 14.9 minutes. Fluorescence detection wasperformed with an excitation wavelength of 320 nm and an emissionwavelength of 420 nm.

Results:

TABLE 2 Lots of D′D3-FP provided for PK analysis: Mono- Di- Tri- Tetra-Asialo sialo sialo sialo sialo Sialylation Lot # [%] [%] [%] [%] [%] [%]B-140526 (no 59.4 29.0 9.7 1.9 n.d. 40.6 temperature shift) B-140616KS16.4 34.6 28.1 15.3 5.6 83.6 B-140825 12.7 42.9 32.0 9.9 2.6 87.3B-140623KS 10.2 38.7 33.8 14.2 3.0 89.8

D′D3-FP protein purified from harvests taken after the temperature shiftfrom 37° C. to 33° C. (e.g. Lot B-140825) or to 34° C. (e.g. LotB-140623KS) showed an improved sialylation pattern in that a reducedamount of asialo and monosialo structures was detected while inparticular the Di-sialo and Tri-sialo structures increased. The reducedcontent of asialo structures had a positive effect on the half-life ofthe D′D3-FP protein itself as well as on a co-administered FVIII (seeexample 8).

A further beneficial effect was found as a result of the temperatureshift in that the ratio of D′D3-FP dimers increased over the monomer atlower temperatures, wherein the dimer is the preferred structure due toits increased binding to FVIII.

TABLE 3 Effect of Temperature on Dimer Content Bioreactor Ratiotemperature before Dimer: harvest % Dimer % Monomer Monomer 37 52.3 47.71.1 35 71.0 29.0 2.45 33 71.2 28.8 2.5 32 74.6 25.4 2.94 31 77.5 22.53.44

As shown in Table 4 the beneficial effect of a temperature shift on thedegree of sialylation was not observed with respect to full length VWF.Specifically, the content of asialostructures could not be reduced whenfull length wild-type VWF albumin fusion (“rVWF-FP”) was expressed undersimilar bioreactor conditions to those described in example 4 and whenthe temperature was reduced to 33.5° C. compared to the expression atthe standard temperature of 37° C. Purification had been performed asdescribed in US 2014/0072561 A1.

TABLE 4 Sialylation of full length VWF Lot # Sialylation rVWF-FP onlyexpressed at 100 37° C. rVWF-FP first expressed at 91% of thesialylation 37° C. then at 33.5° C. degree of rVWF-FP above which wasonly expressed at 37° C.

The degree of sialylation of the Lot harvested at 37° C. was normalisedto a nominal value of 100. The degree of sialylation determined for theLot harvested at 33.5° C. was lower than that of the Lot harvested at37° C.

Example 7: Determination of D′D3-FP Antigen Concentration

Human albumin was determined by an ELISA whose performance is known tothose skilled in the art. Briefly, microplates were incubated with 100μL per well of the capture antibody (goat anti-human-albumin-IgG, Cat.No. A80-129A, Bethyl Laboratories, Inc.), diluted to 2 μg/mL in Buffer A[Sigma C3041] for 16 hours at ambient temperature. After washing platesthree times with buffer B (Sigma P3563), microplates were blocked with200 μL per well of blocking solution (Cat.No. 110500, Candor BiosienceGmbH), for 1.5 hours at ambient temperature. After washing plates againthree times with buffer B (Sigma P3563), serial dilutions of the testsample in LowCross Buffer (Cat. No. 100500, Candor Biosience GmbH,) aswell as serial dilutions of N Protein Standard SL (OQIM13, SiemensHealthcare 50-0.78 ng/mL) in LowCross Buffer (volumes per well: 100 μL)were incubated for one hour at +37° C. After four wash steps with bufferB, 100 μL of a 1:40,000 dilution in blocking solution of the detectionantibody (goat-anti-Human Albumin-IgG peroxidase labelled, Cat. No.A80-129P, Bethyl Laboratories, Inc.)-D, were added to each well andincubated for 45 min. at +37° C. After three wash steps with buffer B,100 μL of substrate solution (1:10 (v/v) TMB OUVF : TMB Buffer OUVG,Siemens Healthcare) were added per well and incubated for 20 minutes atambient temperature in the dark. Addition of 100 μL stop solution (OSFA,Siemens Healthcare) prepared the samples for reading in a suitablemicroplate reader at 450 nm wavelength. Concentrations of test sampleswere then calculated using the standard curve with the N ProteinStandard SL as reference.

Example 8: PK Analysis

Aim

We aimed at characterizing the impact of sialylation on pharmacokinetics(PK) of the half-life extended von Willebrand Factor (VWF) fragmentD′D3-FP dimer and FVIII. One aim of these studies was to determine theinfluence of sialylation of the D′D3-FP dimer on its PK and additionallyon the PK of co-administered FVIII in rats (example 8.1). A secondexample covers the effect on a full-length FVIII product Advate® in rats(example 8.2). The lot # (see Table 2 above) and the degree of D′D3-FPdimer sialylation in % are indicated for each preparation.

Example 8.1: Prolongation of Pharmacokinetics of FVIII byCo-Administration of Highly Sialylated D′D3-FP Dimer in Rats

Material and Methods

Animals: Female Crl:CD (Sprague Dawley) rats in a weight range of230-300 g were breed at Charles River Laboratories (Sulzfeld, Germany).In house, the animals were kept at standard housing conditions, i.e. at21-22° C. under a 12 h/12 h light-darkness cycle. Animals were fed adlibitum with standard rat diet (Ssniff-Versuchsdiäten, Soest, Germany).Tap water was supplied ad libitum. Animal husbandry and study procedurescomplied with the German Animal Welfare law and European Unionregulations.

Laboratory evaluations: The test articles were administered i.v. by asingle injection into the lateral tail vein at a volume of 3 mL/kg. AllD′D3-FP dimer preparations were administered at a dose level of 1000μg/kg based on human albumin values, and co-administered with 200 IU/kgrVIII-SingleChain (rVIII-SC, chromogenic activity) after incubating forapproximately 30 minutes at +37° C. Animals receiving only rVIII-SCserved as control (Table 5).

Blood samples were taken retro-orbitally under short term anaesthesia at5 min, 2, 4, 8, 24, 32, 48 and 72 h after intravenous bolus injectionusing an alternating sampling scheme. The PK profile was taken from twocohorts of rats per group (n=3 per time-point, n=6 per group). Bloodsamples were anticoagulated using sodium citrate (2 parts sodium citrate3.13% +8 parts blood), processed to plasma and stored at −20° C. for thedetermination of FVIII antigen and/or albumin.

D′D3-FP dimer exposure was determined by measurement of the albumin partof the protein using an immunoassay specific for human albumin (example7), and FVIII:Ag plasma levels were detected with the FVIII AsserachromELISA test kit from Stago, S.A.S., France.

TABLE 5 Treatment groups Sialylation [%] D′D3-FP dimer dose FVIII doseTreatment* of D′D3-FP [mg albumin/kg] [IU FVIII:C/kg] rVIII-SC — 200D′D3-FP dimer (B-140526) & rVIII-SC 40.6 1 200 D′D3-FP dimer(B-140616KS) & rVIII-SC 83.6 1 200 D′D3-FP dimer (B-140623KS) & rVIII-SC89.8 1 200 FVIII:C = chromogenic FVIII activity *Lot # given in brackets

Results

D′D3-FP dimer was quantified via its albumin component, and measurementswere performed up to 72 h p.a., and all measured data were well abovethe detection limit of the assay. Mean residence time (MRT) andclearance (CL) were estimated by non-compartmental methods and the dataare presented in FIG. 1. rVIII-SC co-administered with the D′D3-FP dimerwith 40.6% sialylation (B-140526) had a shorter MRT and higher clearanceas when co-administered with the D′D3-FP dimer preparations with 83.6%and 89.8% sialylation (B-140616KS and B-140623KS, respectively).

In line with this observation, the pharmacokinetic profile of theco-administered FVIII (200 IU/kg chromogenic FVIII activity), quantifiedas FVIII:Ag via ELISA, was modified accordingly. It shall be mentionedthat not all plasma levels at 48h and 72 h could be measured, somevalues were below the detection limit of 57 mIU/mL. Clearly, rVIII-SCalone had the shortest MRT and highest clearance, which was generallyprolonged when D′D3-FP dimer was co-administered (FIG. 2). Those D′D3-FPdimers, which had a longer exposure by themselves, also prolonged theFVIII PK profile. Thus, MRT of the D′D3-FP dimer with 40.6% sialylation(B-140526) was shorter and clearance was higher compared to D′D3-FPdimer with sialylation >80%.

Thus, the pharmacokinetic profile of FVIII:Ag was dependent on thesialylation of D′D3-FP dimer, i.e., shortest PK was observed with 40.6%sialylation and longest PK with those of >80% sialylation.

Evaluation of PK characteristics of D′D3-FP dimer was done in moredetail, i.e. additionally calculating maximal concentrations (C_(max))and terminal half-life (t½) in a non-compartmental model, as well ascalculating the x-fold increases (Table 6). Sialylation between 89.8%and 40.6% influenced clearance of D′D3-FP dimer by more than 2-fold(0.91 mUkg/h for the 89.8% D′D3-FP dimer and 2.06 mL/kg/h for the 40.6%D′D3-FP dimer as determined by measuring the albumin concentration overtime). This relates to more than 40% increase in mean residence time(MRT, i.e. 56.9 h to 81.5 h) and more than 30% increase in terminalhalf-life (i.e. 44.0 h to 58.6 h).

As depicted in the graphs for MRT and clearance, this translates to thePK characteristics of the co-administered FVIII, even though not asobvious as for D′D3-FP dimer (Table 6, FVIII:Ag): clearance is decreasedby more than 30% (3.93 mL/kg/h to 2.95 mL/kg/h), MRT is increased by 19%(16.5 h to 19.6 h) and terminal half-life by 15% (11.4 h to 13.1 h).

Therewith, the increase in exposure over time is given by D′D3-FP dimerdepending on the percentage of sialylation, as may also be seen by thefold increase of PK characteristics of rVIII-SC given alone. While 40.6%sialylation prolong FVIII PK 1.5-1.9 fold, an optimized D′D3-FP dimerwith 89.8% sialylation prolongs FVIII PK 2.0-2.2 fold, and 83.6%sialylation leads to intermediate values. Thus, this effect correlateswith the degree of sialylation within the investigated range from 40.6%to 89.8%.

TABLE 6 Pharmacokinetic parameters of D′D3-FP dimer and FVIII: Ag afterco-administration of rVIII-SC and D′D3- FP dimer in rats(non-compartmental analysis) Dose D′D3-FP dimer 1 mg/kg, dose rVIII-SC200 IU/kg Half-life, C_(max), extrap. Clearance MRT terminal Treatment*IU/mL mL/kg/h h h Albumin D′D3-FP dimer (40.6%) & 18.1 2.06 56.9 44.0rVIII-SC D′D3-FP dimer (83.6%) & 18.8 1.07 82.4 61.4 rVIII-SC D′D3-FPdimer (89.8%) & 21.3 0.91 81.5 58.6 rVIII-SC FVIII:Ag rVIII-SC 4.26 6.048.9 6.4 D′D3-FP dimer (40.6%) & 3.05 3.93 16.5 11.4 rVIII-SC 1.5fold1.9fold 1.8fold D′D3-FP dimer (83.6%) & 3.41 3.41 18.1 12.8 rVIII-SC1.8fold 2.0fold 2.0fold D′D3-FP dimer (89.8%) & 3.97 2.95 19.6 13.1rVIII-SC 2.0fold 2.2fold 2.0fold *degree of D′D3-FP dimer sialylationgiven in brackets

Example 8.2: Prolongation of Pharmacokinetics of Full-Length FVIII byCo-Administration of Highly Sialylated D′D3-FP Dimer in Rats

Material and Methods

Animals: Female Crl:CD (Sprague Dawley) rats in a weight range of220-300 g were breed at Charles River Laboratories (Sulzfeld, Germany).In house, the animals were kept at standard housing conditions, i.e. at21-22° C. under a 12 h/12 h light-darkness cycle. Animals were fed adlibitum with standard rat diet (Ssniff-Versuchsdiäten, Soest, Germany).Tap water was supplied ad libitum. Animal husbandry and study procedurescomplied with the German Animal Welfare law and European Unionregulations.

Laboratory evaluations: The test articles were administered i.v. by asingle injection into the lateral tail vein at a volume of 3 mL/kg. AllD′D3-FP dimer preparations were administered at a dose level of 1000μg/kg based on human albumin values, and co-administered with 200 IU/kgAdvate® (nominal chromogenic activity) after incubating forapproximately 30 minutes at +37° C. Animals receiving only Advate®served as control (Table 7).

Blood samples were taken retro-orbitally under short term anaesthesia at5 min, 2, 4, 8, 24, 32, 48 and 72 h after intravenous bolus injectionusing an alternating sampling scheme. The PK profile was taken from twocohorts of rats per group (n=3 per time-point, n=6 per group). Bloodsamples were anticoagulated using sodium citrate (2 parts sodium citrate3.13%+8 parts blood), processed to plasma and stored at −20° C. for thedetermination of FVIII antigen and/or albumin.

D′D3-FP dimer exposure was determined by measurement of the albumin partof the protein using an immunoassay specific for human albumin (example7), and FVIII:Ag plasma levels were detected with the FVIII AsserachromELISA testkit from Stago, S.A.S., France.

TABLE 7 Treatment groups (experiment-wise) Sialylation D′D3-FP dimerdose FVIII dose Treatment* [%] [mg albumin/kg] [IU FVIII:C/kg] Advate ®— 200 D′D3-FP dimer (B- 40.6% 1 200 140526) & Advate ® D′D3-FP dimer (B-87.3% 1 200 140825) & Advate ® FVIII:C = chromogenic FVIII activity *Lot# given in brackets

Results

D′D3-FP dimer was quantified via its albumin component, and measurementswere performed up to 72 h p.a., and measured data were well above thedetection limit over the whole observation period. Mean residence time(MRT) and clearance (CL) were estimated by non-compartmental methods andthe data are presented in FIG. 3. PK characteristics of D′D3-FP dimer inthe group of Advate® co-administered with the D′D3-FP dimer with 40.6%sialylation had a shorter MRT and higher clearance as whenco-administered with the D′D3-FP dimer preparation with 87.3%sialylation.

In line with this observation, the pharmacokinetic profile of theco-administered FVIII (200 IU/kg nominal chromogenic FVIII activity),quantified as FVIII:Ag via ELISA, was modified accordingly. It shall bementioned that samples could be measured until 4-8 h p.a. with theAdvate-treated group and until 24-32 h p.a. with the D′D3-FP dimerco-treated groups, thereafter the values were below the limit ofdetection of the assay of 117 mIU/mL. Clearly, Advate® alone had theshortest MRT and highest clearance, which was generally prolonged whenD′D3-FP dimer was co-administered (FIG. 4). Those D′D3-FP dimers, whichhad a longer exposure by themselves, also prolonged the FVIII PKprofile. Thus, MRT of the D′D3-FP dimer with 40.6% sialylation wasshorter and clearance was higher compared to D′D3-FP dimer withsialylation >85%. Thus, the pharmacokinetic profile of FVIII:Ag wasdependent on the sialylation of D′D3-FP dimer, i.e., shortest PK wasobserved with 40.6% sialylation and longest PK with those of >85%sialylation.

Evaluation of PK characteristics of D′D3-FP dimer was done in moredetail, i.e. additionally calculating maximal concentrations (C_(max))and terminal half-life (t½) in a non-compartmental model, as well ascalculating the x-fold increases over Advate® given alone (Table 8).

Sialylation between 87.3% and 40.6% influenced clearance of D′D3-FPdimer by more than 1.5-fold (1.32 mL/kg/h for the 87.3% D′D3-FP dimerand 2.17 mL/kg/h for the 40.6% D′D3-FP dimer as determined by measuringthe albumin concentration over time). This relates to slight effects onmean residence time (MRT, +14%, i.e. 54.4 h to 62.0 h) and terminalhalf-life (t½, +4%, i.e. 42.2 h to 44.0 h).

As depicted in the graphs for MRT and clearance, this translates to thePK characteristics of the co-administered FVIII, even though mostly notas obvious as for D′D3-FP dimer (Table 8, FVIII:Ag): clearance isdecreased by more than 20% (12.99 mL/kg/h to 10.66 mL/kg/h), MRT isincreased by 12% (10.2 h to 11.4 h) and terminal half-life by 11% (8.9 hto 9.9 h).

Therewith, also for the full-length FVIII product Advate®, the increasein exposure over time is given by D′D3-FP dimer depending on thepercentage of sialylation, as may also be seen by the fold increase ofPK characteristics of Advate® given alone. While 40.6% sialylationprolong FVIII PK 2.3-2.9fold, an optimized D′D3-FP dimer with 87.3%sialylation prolongs FVIII PK 2.8-3.2fold.

TABLE 8 Pharmacokinetic parameters of D′D3-FP dimer and FVIII: Ag aftercoadministration of Advate ® and D′D3-FP dimer in rats(non-compartmental analysis) Dose D′D3-FP dimer 1 mg/kg, dose Advate ®200 IU/kg Half-life, C_(max), extrap. Clearance MRT terminal Treatment*IU/mL mL/kg/h h h Albumin D′D3-FP dimer (40.6%) & 18.8 2.17 54.4 42.2Advate ® D′D3-FP dimer (87.3%) & 17.2 1.32 62.0 44.0 Advate ® FVIII:AgAdvate ® 3.29 29.55 3.5 3.1 D′D3-FP dimer (40.6%) & 3.19 12.99 10.2 8.9Advate ® 2.3fold 2.9fold 2.9fold D′D3-FP dimer (87.3%) & 3.38 10.66 11.49.9 Advate ® 2.8fold 3.3fold 3.2fold *degree of D′D3-FP dimersialylation given in brackets

Conclusion from PK Study Results

These studies demonstrate that co-administration of D′D3-FP dimer andFVIII prolongs FVIII:Ag plasma exposure using different FVIII products.This prolongation is dependent on the status of sialylation of D′D3-FPdimer: generally, a better sialylation further optimizes FVIII plasmaexposure. In detail, D′D3-FP dimer with a sialylation of 40.9% wasinferior with regard to FVIII:Ag plasma exposure to D′D3-FP withsialylation in the range of 83.6-89.8%.

Since in the rat (in contrast to human haemophilia A patients), humanand endogenous FVIII compete with D′D3-FP dimer binding sites, it may beexpected that the effect on FVIII in the human haemophilia patient iseven stronger.

Example 9: In Vitro Sialylation of D′D3-FP

D′D3-FP dimer was dialyzed against 35 mM sodium acetate/35 mM Trisbuffer at pH 7.0. To about 600 μg of the protein in 110 μl, 0.75 mgCMP-NANA (Roche Cat.No 05974003103) dissolved in 100 μl water as donorsubstrate and 10.5 μl ST6GAL-1 (60 μg, Roche Cat.No 07012250103, inwater) were added. The mixture was incubated at 37° C. for 6 hours andthe reaction was stopped by freezing at −15° C. to −25° C. Thisprocedure was according to the manufacturer's recommendation. D′D3-FPdimer was then purified from the reagents by chromatography using SECSuperdex 200 pg (GE Healthcare, Code 90-1002-10). Sialylation wasdetermined as described above and the results are given in Table 9.

TABLE 9 results of an in vitro sialylation study Lot # Sialylationstarting material 100 after in vitro 137% of the sialylation degree ascompared sialylation to the starting material

The degree of sialylation of the starting material was normalised to anominal value of 100. The degree of sialylation after in vitrosialylation was substantially higher than that of the starting material.

Example 10: Anion-Exchange Chromatography to Enrich for HighlySialylated VWF Fragments

D′D3-FP prepared according to example 5 is further purified using anionexchange chromatography to reduce the content of asialo N-glycanstructures. Therefore, the D′D3-FP solution is diluted using 20 mMTris×HCl pH 7.4 buffer to a conductivity low enough to allow completebinding of D′D3-FP to the column (in general below 5 mS/cm) and loadedon a chromatography column (fill height approximately 20 cm) filled withPoros XQ resin that was equilibrated using equilibration buffercontaining 20 mM Tris×HCl, 20 mM NaCl pH 7.4. After washing the columnwith equilibration buffer, D′D3-FP is eluted using a flat lineargradient from equilibration buffer to elution buffer (20 mM Tris×HCl,500 mM NaCl pH 7.4). The elution peak containing D′D3-FP is fractionatedinto approximately 10 fractions of similar volumes and the early peakfractions that contain D′D3-FP with increased amounts of asialo N-glycanstructures are discarded and the later peak fractions containing asialoN-glycan structures below the desired level (e.g. 20% or lower) arepooled.

Alternatively the purification run of D′D3-FP is performed with thedifference that pooling of D′D3-FP eluate peak fractions is only donefor those fractions containing D′D3-FP with an asialo N-glycan structurecontent of below 15% (or below 10%).

As described, by pooling of corresponding fractions suitable D′D3-FPpreparations can be manufactured with a desired maximum content ofasialo N-glycan structures.

Based on the results obtained with a linear gradient used for elution,step gradients with buffers containing different concentrations of NaClcan be derived that also allow removal of first fractions with higheramounts of asialo N-glycan structures thus resulting in D′D3-FP eluateswith content of below 15% of asialo N-glycan structures.

Example 11: Determination of FVIII Affinity to VWF Fragment Dimer andMonomer

D′D3-FP monomer and dimer were isolated as described above, and theaffinity of FVIII to these preparations was assessed through surfaceplasmon resonance via a Biacore instrument (T200, GE Healthcare).

An anti-albumin antibody (MA1-20124, Thermo Scientific) was covalentlycoupled via its N-terminus to an activated CM 3 chip by NHS(N-Hydroxysuccinimide) and EDC (Ethanolamine hydrochloride), bothcontained in the amine coupling kit (BR1000-50) from GE Healthcare. Forimmobilization 3 μg/mL of the antibody were diluted in sodium acetatebuffer (10 mM, pH 5.0) and the antibody solution was flown over the chipfor 7 min. at a flow rate of 10 μL/min. After the immobilizationprocedure non-coupled dextran filaments were saturated by flowingethanolamine solution (1 M, pH 8.3) over the chip for 5 min (at a flowrate of 10 μL/min). The aim of saturating the flow cell was to minimizeunspecific binding of the analytes to the chip. A reference flow cellwas set up by saturating an empty flow cell with ethanolamine by usingthe same procedure as above.

Dimeric and monomeric D′D3-FP proteins, respectively, were immobilizedto the covalently coupled anti-albumin antibody by a flow of the D′D3-FPproteins (5 μg/mL) over the chip for 3 min (flow rate of 10 μL/min). Thecaptured mass of dimeric D′D3-FP was 335 RU and for monomeric D′D3-FP147 RU, assuming one binding site both on the monomer and on the dimerD′D3-FP for FVIII.

To create binding curves for FVIII, each D′D3-FP protein preparation wasdiluted in running buffer (HBS-P+: 0.1 M HEPES, 1.5 M NaCl and 0.5% v/vSurfactant P20, pH 7.4; product code BR100671, GE Healthcare) toconcentrations of 0.25 nM, 0.5 nM, 1 nM, 3 nM and 4 nM. By performing asingle cycle kinetic, samples with ascending concentrations of eachdilution were flown over the chip for 2 min (flow rate 30 μL/min.),followed by a dissociation time of 10 min. with running buffer HBS-P+.All measurements were performed twice. The temperature for the measuringprocedure was adjusted to +25° C.

Binding parameters were calculated using BiaEvaluation Software. Thecurve fitting methods were based on Langmuir equations. The input datafor calculations were the molar mass of the analyte FVIII(rVIII-SingleChain) of 170 kDa, other parameters like max. RU and slopeswere automatically extracted out of the fitted association anddissociation curves. The outputs of BiaEvaluation Software are theassociation rate constants and the dissociation rate constants, fromwhich the affinity constants were calculated. The results are shown inTable 10.

TABLE 10 FVIII affinity data for D′D3-FP dimer and monomer D′D3-FPpreparation ka [1/Ms] kd [1/s] KD [M] D′D3-FP Dimer 2.33E+07 1.37E−035.90E−11 D′D3-FP Monomer 4.41E+07 3.96E−03 8.99E−11

The association rate constant was slightly increased forrVIII-SingleChain to the monomeric D′D3-FP, while the dissociation rateconstant of rVIII-SingleChain to D′D3-FP dimer was three times slowerthan to the monomer. The quotient of the dissociation rate constant andthe association rate constant indicates the affinity ofrVIII-SingleChain to D′D3-FP. The dimeric D′D3-FP hence shows anincreased affinity to FVIII compared to the D′D3-FP monomer.

Example 12: Quantitative Determination of Individual N-Glycan Species

Percentage of all N- Percentage of all N- glycans with two or glycanswith three or more terminal and more terminal and non-sialylatedgalactose non-sialylated galactose residues residues Lot # [% of totalN-glycans] [% of total N-glycans] B-140526 (no 38.7 6.9 temperatureshift) B-140616KS 21.2 2.2 B-140825 17.8 1.9 B-140623KS 9.5 1.1

The N-Glycans released by PNGase F were labelled with a fluorophore2-aminobenzamide (AB) and purified prior analysis using in-lineLC-fluorescence—high resolution MS detection allowing simultaneousquantitative determination and identification of the labelled N-Glycansusing accurate mass and retention time information. Using a mixed modeHILIC/RP LC-column allowed the separation of the released and ABlabelled N-Glycans based on charge and structure which enabled aquantitative determination of different structures according to thenumber of terminal galactose and non-sialylated residues. The standarddeviation of the fluorescence quantitation using the area under curvewas found to be on average less than 0.5% using a reference sample(n=5). The presence of terminal and non-sialylated galactose residues inthe separated AB labelled N-glycans was confirmed by treating thereleased AB-labelled N-glycans with β1-4-Galactosidase and re-injectingthem using the same LC-FLD-MS methods and analysing the shifted peaks.

The Following Methods Were Applied:

PNGase F Enzymatic Glycan Release:

About 700 μg of the purified protein was reduced with DTT in ammoniumbicarbonate, pH 8.5 at 60° C. for 30 min. The reduced sample was cooledto room temperature and alkylated with iodoacetamide at RT in the darkfor 30 min. The alkylated sample was buffer exchanged into 50 mMammonium bicarbonate pH 8.6 using a 2 mL Zeba Spin 7K MWCO column. Tothe buffer exchanged sample, 40U of PNGase was added and the sampleincubated at 37° C. for 14 hours. An additional 40U of PNGase was addedand the sample incubated for a further 6 hours at 37° C. . The PNGasedigested sample was centrifuged through a 50KDa Amicon Ultra 4ultrafilter. The filtrate was dried in a CentriVap.

2-AB Labelling of Released N-Glycans:

The 2-AB labelling reagent was prepared following the manufactureinstructions. 50 μL of the 2-AB reagent was added to the dried sampleand incubated in the dark at 65° C. for 3.5 hours.

A Waters Oasis HLB 3 cc 60 mg SPE cartridge was conditioned with 3 mL95% acetonitrile the 3 mL 35% acetonitrile then 3 mL 95% acetonitrile.The 2-AB labelled sample was diluted by adding 1.95 mL of 95% v/vacetonitrile and immediately loaded onto the HLB cartridge and allowedto drain under gravity. Sample was washed under gravity with 3×3mL of95% v/v acetonitrile and the eluted with 3 mL of 35% v/v acetonitrile.The dried 2-AB derivatised sample was dissolved by the addition of 35 μLof Milli Q water and vortex mixing. After dissolution, 85 μL ofacetonitrile was added and mixed briefly. The sample was transferred toa HPLC vial for analysis.

2-AB Glycan Analysis:

High performance liquid chromatography was performed on a Thermo DionexUltimate 3000 system consisting of an RS Binary Pump, Autosampler , RSColumn Compartment and RS Fluorescence detector. The separation of 2-ABglycan derivatives was achieved using a Dionex GlycanPac AXH-1, 1.9 μm,2.1×150 mm column (P/N 082472). Mobile phase A consisted of 100%acetonitrile, Mobile phase B consisted of 50 mM formic acid adjusted topH 4.0 with 5M ammonium hydroxide solution. The column was maintained at50° C. and the flow rate was 0.200 mL/min. Fluorescence detection wasperformed with an excitation wavelength of 320 nm and an emissionwavelength of 420 nm.

The LC-FLD system was coupled to a high resolution orthogonal TOF-MS(MaXis, Bruker-Daltonik, Bremen, Germany). The transfer capillary waskept at a voltage of −4500 V (positive ion polarity mode). The nebulizerwas set to 0.8 bar using the standard ESI sprayer (Bruker, Bremen,Germany), the dry gas temperature to 180° C. and the dry gas flow-rateto 7 L/min. The ion transfer was optimized in the range m/z 200-3000 forhighest sensitivity while keeping the resolution R>50,000 across thewhole mass range. The TOF-MS mass calibration was carried out prior theLC-MS experiment by direct infusion of a 100 fold dilution of ES TuningMix (Agilent Technologies, Waldbronn, Germany) at 4 ul/min.

1.-21. (canceled)
 22. A method of treating a blood coagulation disorder,comprising administering to a subject in need thereof an effectiveamount of a glycoprotein and an effective amount of a Factor VIII(FVIII), wherein the glycoprotein comprises a truncated von WillebrandFactor (VWF) capable of binding to the FVIII, wherein the glycoproteincomprises N-glycans, wherein i) at least 75% of the N-glycans compriseat least one sialic acid moiety, ii) less than 35% of the N-glycanscomprise two or more terminal and non-sialylated galactose residues,and/or iii) less than 6% of the N-glycans comprise three or moreterminal and non-sialylated galactose residues, wherein the glycoproteinis administered intravenously or subcutaneously, and wherein the FVIIIis administered intravenously or subcutaneously.
 23. The methodaccording to claim 22, wherein the mean residence time (MRT) of theFVIII is increased and/or the clearance of the FVIII is decreased byco-administration of the glycoprotein, as compared to a treatment withFVIII alone; and/or wherein the frequency of administration of the FVIIIis reduced as compared to a treatment with FVIII alone.
 24. The methodof claim 22, wherein the glycoprotein and the FVIII are administeredsimultaneously.
 25. The method of claim 22, wherein the glycoprotein andthe FVIII are administered separately and sequentially.
 26. The methodof claim 22, wherein at least 70% of the N-glycans comprise at least oneα-2,6-sialic acid moiety or at least one α-2,3-sialic acid moiety. 27.The method of claim 22, wherein at least 80% of the N-glycans compriseat least one sialic acid moiety.
 28. The method of claim 22, wherein atleast 85% of the N-glycans comprise at least one sialic acid moiety. 29.The method of claim 22, wherein the truncated VWF comprises (a) aminoacids 776 to 805 of SEQ ID NO:9, or (b) an amino acid sequence having atleast 90% sequence identity to amino acids 776 to 805 of SEQ ID NO:9.30. The method of claim 22, wherein the truncated VWF consists of (a)amino acids 776 to 805 of SEQ ID NO:9, or (b) an amino acid sequencehaving at least 90% sequence identity to amino acids 764 to 1242 of SEQID NO:9
 31. The method of claim 22, wherein the truncated VWF comprises(a) amino acids 764 to 1242 of SEQ ID NO:9, or (b) an amino acidsequence having at least 90% sequence identity to amino acids 764 to1242 of SEQ ID NO:9.
 32. The method of claim 22, wherein the truncatedVWF consists of (a) amino acids 764 to 1242 of SEQ ID NO:9, or (b) anamino acid sequence having at least 90% sequence identity to amino acids764 to 1242 of SEQ ID NO:9.
 33. The method of claim 22, wherein ahalf-life extending heterologous polypeptide is fused to the truncatedVWF, and/or a half-life extending moiety is conjugated to theglycoprotein.
 34. The method of claim 22, wherein the glycoprotein is adimer.
 35. The method of claim 34, wherein the affinity of the dimericglycoprotein to FVIII is greater than the affinity of the correspondingmonomeric glycoprotein to FVIII.